Udaat Mittu

Replying to the comments of peer review: 1. Combinations of keywords have been used to avoid going into application based studies and clinical trials. Data regarding one word keywords vs combined keywords had been given in a previous assignment. The combinatorial keywords allow for more speci c hits, keep the review focused on the technique and its major applications rather than going awry. Hence, such complex keywords were used out of necessity rather than choice given than the amount of literature that exists under the banner of CRISPR/Cas9. Combination keywords have also been used because it was observed that searches for a complex keyword retrieved hits which had partial identity to the keyword. An example of this would be using the keyword Cas9 Gene editing. If a speci c search for Cas9 gene editing is made, the review will pop up early due to complete identity with the keyword. However, if a non-speci c search using the query ‘gene editing’ is made, the review will still pop up, albeit a bit later. This would not happen in the non-speci c case if the keyword had just been Cas9 or gene editing. Thus, using complex keywords would allow a speci c search to nd the review early, yet will not completely disqualify the review from popping up due to partial identity between keywords and the search query. The keywords were designed keeping that cyber trick in mind, thus, they have not been changed. 2. No changes to the abstract have been made as the author feels it has enough information already and the word limit must be strictly adhered to. The abstract was kept playful intentionally. The technique is talked about as a genetic editing technique, and the fact that it has variants is mentioned. Further, the criticism that the system of CRISPR must be de ned seems a bit misplaced. CRISPR has no given system. It works in many systems and stems from multiple bacteria. Even its discovery included multiple systems. Hence de ning a basic CRISPR ‘system’ is not possible. 3. All full forms have already been mentioned in the text. Cpf1 has no full form. KRAB full form has been added. 4. Speci c grammatical and syntax errors have been corrected. 5. More schematics have been added where appropriate. Explanations have been provided in the gure legends as well. 6. APOBEC1 is APOBEC1. A search for APOBAC1 yields APOBEC1 as a suggestion by google. It is thus a suggestion to the reviewer to please make such comments carefully and after conducting proper research. 7. The manuscript has been kept as brief as possible. Nuances have been avoided multiple times in order to not go completely away from the main point of the review. fi fi fi fi fi fi fi fi fi fi 1 Seeing as how the purpose of the review is to serve as a hub of all things related to CRISPR, such length is unavoidable as the scope of the review is extremely wide. Such a criticism is understandable given that in the basics of CRISPR section, a lot of details about the functioning of the enzyme have been given, but given the fact that the review wants the reader to understand CRISPR at a satisfactory, it is necessary that such details are provided, otherwise, the reader might get a false sense of the true exibility CRISPR/Cas9 application. Also, the details mentioned in this section have been used in other sections to explain follow-up concepts, hence no details have been removed. 8. A lot of subheadings have been used to cover the vast area under the banner of CRISPR/Cas9 application. Each subheading covers a major application of the technique. Thus, a reduction in their number would mean blatantly ignoring the existence of a major part of the technique and its future, hence taking away from the reason behind the review, which is to provide a ‘COMPREHENSIVE’ look at the technique in its entirety. Thus, no sub-headings have been removed. 9. Finally, colourful fonts have been chosen in order to di erentiate between the various kinds of headings. Major headings, section headings, section sub-headings, subheadings under section sub-headings, all have been provided a di erent colour so that the reader is not lost when they read a heading. It is therefore a request to the reviewer then to please understand the reason behind the colours and not just criticise their use in general. Such colours also introduce visual variety in a text which is long and contains many details. Colours have been accepted as a part of schematics, it is time we adapt them into the main text as well, as a visual treat to the eyes. Thus, keeping all that in mind, the colour scheme has been kept unchanged. 10. References were checked and were deemed appropriate. Citations in the main text have been xed. 11. An author’s note: Most of this review has been written at the level of an undergraduate student or a student who is currently beginning their graduation. It is for this very reason that casual and playful language is used and nuances have been avoided to keep the review from getting too technical and complicated. The review expressly mentions where nuances have been avoided so that readers are not completely blindsided. Thus, it aims to be more of a gentle read rather than a technical scienti c text. It is therefore requested that the reader read through the review in that eye rather than judging this text on the same merits as that of a formal scienti c text. To achieve such a review has been a painstaking endeavour. A balancing act of what to include and what not to include. Almost all major uses of the technique have been covered and all major new developments have been covered as well, thus the review ends up becoming a long read. To avoid having the reader go through all of it at once, the review has been cleanly divided into three major sections. The introduction lists what all each section includes so that the reader can jump directly to that section and hence have quick access to the information that they need. This is another reason that the colour scheme is being kept, so that the reader knows when the jump to another major section is occurring. A mini conclusion has been added as a cool down after the read. ff ff fi fi fi fl 2 CRISPR/Cas9 system: Origins, the present scenario and beyond By Mittu Udaat In 2002, Ruud Jansen and colleagues coined the term CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) for a sequence of direct repeats interspaced with non-repetitive spacers. What followed in the next 2 decades has turned the CRISPR/Cas system into one of the most exible and widely used techniques in Biological research and has brought about a revolution in the eld of genetic editing. It has expanded far beyond that with variants coming out every year which makes keeping track of the latest developments of this tool di cult. The aim of this review thus, is to serve as a hub of everything related to the CRISPR/Cas9 system, be it historical perspective, major variants in use today or newer versions and improvements to the technique popping up every day. Hence, anyone looking to familiarise themselves with the technique has quick access to all things CRISPR via this review. Keywords: Clustered regularly interspaced short palindromic repeats, CRISPRassociated proteins, CRISPR-Cas systems, Cas9 Gene editing, CRISPR-associated protein 9, CRISPR Introduction: fi fi fi fi fi fl ffi fl fi fi CRISPR/Cas9 system is a technology that was developed by Jennifer Doudna and Emmanuelle Charpentier in 2012 as a method of targeted gene editing. (18) Since its development, this technology has created waves of scienti c applications and has generated myriads of variants. (36) Such has been its impact that almost every Biological science lab in the world seems to have adopted it in some manner. It is thus important for any person who wants to conduct research in this eld to be aware of the existence of this technique, have a general idea of its mechanism of function and possess some knowledge of the many ways in which it is being applied in modern scienti c research. To aid in that, this review tells the story of how CRISPR/Cas9 system was found and how it was turned into a tool, how it functions (the molecular mechanism) and this is followed by an exploration of the major applications of the technique today. As a treat for those who are interested, a section discussing the future of CRISPR/Cas9 has been included which discusses the latest phenomenons in the evolution of CRISPR/Cas9 technology. All of this is underlined by a nal section which talks about the human side of science, the ethics of using a gene editing technology. Thus, this review consists of three separate sections: 1. The Origins: A section which talks about the history of development of CRISPR/Cas9 2. The Present Scenario: A section which explains the mechanism of action of the method and covers the major applications of CRISPR/Cas9 system. 3. Beyond: The nal section which talks about the interesting experiments that have been conducted in order to push this technique even further. Articles have been discussed which describe how major obstacles limiting the universal application of this technology are being tackled. This section also discusses the ethics of use of such a powerful and exible technology. It is requested of the reader to pick a section of their choice from the three described above based on what information they would like to absorb rst and what their purpose is 3 for reading this review. For that very reason, the sections have been kept somewhat stand alone. With that said, let's dive into the rst section. The Origins: In this section, we take a look at the journey the scienti c community took in order to create the CRISPR/Cas9 system. Step by step, we shall take a look at the landmark discoveries that happened in the unbelievable ride that scientists experienced on the road to developing the most exible and applicable biotechnological method ever. History of CRISPR: Finding the elusive repeats: In 1987, as a part of an article completely unrelated to the repeats now known as CRISPR, the rst instance of a sequence of repeats interspersed with spacers was mentioned, that too as an afterthought in the ‘Discussion’ section. The article was about a product of the iap gene in Escherichia coli and by accident, regions surrounding the iap gene were sequenced. The discussion mentioned that the locus had regions of homology spaced by 32 nucleotide spacers and that the biological function of such a construct was ‘not known’. (1) It was an obscure beginning for a tool that in a matter of a few decades would go on to dominate the world of biotechnology. After the discovery of the repeats, no major waves were made. The research article became part of an ocean of scienti c literature, with a little pearl hidden in its written word. Fast forward now to 1993, in an early article about spoligotyping for di erentiation of Mycobacterium tuberculosis strains, a mention was made about a sequence of direct repeats with a length of 36 nucleotides. (2) Again, no speci c follow up was made for these repeats. As happens often in science, in the same year, in a completely unrelated scienti c study about the alterations of genetic loci of a microorganism in conditions of varying salt concentrations, Mojica, a graduate student rediscovered the same repeats in Haloferax mediterranei and for the rst time, someone’s interest was sparked by these unknown repeats. In 1993, he published an article describing these palindromic repeats of 30 nucleotides interspersed with short sequences and noted that these repeats were unlike any other. Little did he know at that time, the gravity of the discovery he had made and what a wild re his spark of interest would generate. In follow up works, he noted that these repeats were present in multiple organisms, and by the year 2000, he had characterised these repeats in almost 20 di erent organisms. (3) fi fi fi fi ff fi ff fi fl fi fi fi By this time, thanks to the work of Mojica, a good number of scientists had taken note of this sequence that was then called ‘Short regularly spaced repeats’ (SRSRs) and by the year 2002, it had been discovered that these repeats were present in a large number of organisms and there were dedicated genes (cas genes) present in their vicinity. (4) It was in 2002, that on the suggestion of Mojica, Ruud Jansen and colleagues coined the term ‘Clustered regularly interspaced short palindromic repeats’ for this family of repeats. (4) The article published by Jansen et al. was also one of the rst articles to capture the structure of the entire CRISPR construct. (3) The structure of the CRISPR locus was 4 elucidated. It was time then to gure out, what do these repeats do and why are they dispersed across so many organisms. From repeats to the tool: Spacers are of extrachromosomal origin We arrive at a point now where the various parts of the CRISPR loci have been found, yet their function remains unknown. In 2002, it was still not known what the spacers between the repeats were responsible for, or what they were homologous to. This mystery was solved independently by three players, an old one, Mojica and the new ones Gilles Vergnaud and Alexander Bolotin. Mojica, in order to decipher the mystery behind the spacers, chose the most obvious method. He picked up sequences of these spacers and used the bioinformatic tool BLAST to search for similar sequences. It was in 2005 that he discovered that one of his spacer sequences was similar to a P1 bacteriophage. The curious thing about the strain of the organism with this spacer was that this very organism was also resistant to P1 bacteriophage. Thus, Mojica hypothesised that the loci might have functions similar to the adaptive immune system. (5) Independently of Mojica, Vergnaud studied the spacers present in Yersinia pestis and he discovered that the spacers present in the CRISPR locus of the microbe were derived from a bacteriophage and that these spacers were added in a directional manner such that a new spacer was always added at the front end. (6) Thus, in this manner, two individual scientists came to the conclusion that the CRISPR locus must be responsible for immunity against incoming infections. It was aptly described as ‘memory of past genetic aggressions’. The third player, Alexander Bolotin also arrived at the same conclusion as the formers. What is notable about his work is that Bolotin was the rst person to suggest a possible mechanism of the functioning of CRISPR locus. He suggested that the spacers when transcribed, must work in an antisense inhibition manner, by binding directly to bacteriophage RNA and preventing its proper functioning, in a manner similar to RNAi (RNA interference). (7) CRISPR is responsible for ‘adaptive’ immunity The fact that spacers in the CRISPR loci were of extrachromosomal origin and that they were derived from bacteriophages immediately gave birth to the speculation that the CRISPR locus might be involved in conferring adaptive immunity to a bacterial system. In 2007, a landmark article by Phillipe Horvath and colleagues proved beyond any doubt that CRISPR in-fact led to development of adaptive immunity. Horvath and colleagues used a bacteriophage sensitive Streptococcus thermophilus and two di erent bacteriophages isolated from di erent samples of yogurt. In this article, they showed that resistance to infection was acquired only when a corresponding spacer which was identical to the bacteriophage was present inside the CRISPR locus. Another curious discovery was that an increase in the number of the same spacers would lead to increased resistance. The crux of the article however was that 100% identity between the spacer and the bacteriophage genome was required for proper functioning. Bacteriophages which displayed a single nucleotide polymorphism bypassed the CRISPR adaptive immune system and thus killed the microbes. Similar results were observed for fi ff ff fi 5 spacers which displayed any genetic alterations. The same group also showed that a protein of the Cascade region Csn1 (now Cas9) acted as a nuclease. (8) Thus, by 2007, the exact function of the CRISPR locus was elucidated. The scienti c community then aimed its e orts towards understanding the mechanism of CRISPR adaptive immunity in its entirety by looking at it on the molecular level. The bits and bobs of CRISPR based adaptive immunity In 2008, Van der Oost and colleagues looked to understand how various parts of the CRISPR locus functioned. To do that, they took two Escherichia coli strains, one with a CRISPR locus and one without and transferred the CRISPR locus from the one containing the locus into the strain without one. Then, they knocked out each component of the CRISPR locus one at a time and that led to the discovery of 5 proteins which are now termed ‘Cascade’ proteins and which were named ‘cas’ for CRISPR associated. Cascade proteins were found to be necessary for cleaving a precursor RNA transcribed from the CRISPR locus to form crRNAs (CRISPR RNAs). The crRNAs consisted some of the last nucleotides of the repeat region, the complete spacer and the bases at the beginning of the repeat region which immediately followed the spacer. Such a construction led to secondary structure formation due to complementarity between the repeat elements since they are palindromic. The same group also went on to prove that crRNAs were required for CRISPR based immunity by programming the crRNAs to target the lambda phage. This was the rst time ever that anyone had arti cially programmed a CRISPR locus to speci cally target a gene of interest. (9) Even after all this information was available, it was still not clear whether CRISPR RNA targeted RNA or DNA? According to Bolotin, RNA was the target of interest, but this was refuted by a student by the name of Mara ni. (3) In 2008, Mara ni and Sonheimer used a modi ed plasmid which had a self splicing region in a reporter gene in the plasmid. The experiment was simple. If crRNAs targeted only RNA, no changes would be noticeable since the given region of the reporter plasmid would be spliced out and the rest of the mRNA would be targeted as is normal. But if crRNA attacked DNA, the extra region inserted inside the reporter would be incompatible with the crRNA and thus, CRISPR based immunity would not be observed. Using this method, they proved that crRNAs did target DNA and that CRISPR system was basically a targeted DNA endonuclease. (10) After all this, focus was shifted towards the main mechanism of CRISPR function. By now, it was known that Cas9 functioned as a nuclease (8) and that crRNA targeted DNA. (10) Garneau et al. followed up these studies and went on to then show that CRISPR/Cas in Streptococcus thermophilus produced blunt ended double stranded cuts. The cuts were quickly followed by a conserved sequence of 3 nucleotides called the protospacer adjacent motif. (PAM) (11,12,13,14) This showed that the cuts were made precisely at the regions of complementarity. Another piece to the puzzle of CRISPR function was found by Charpentier and Vogel in 2012. In an independent experiment, they found that the third most expressed RNA in Streptococcus pyogenes was a region from the CRISPR locus which would be called the tracrRNA (Trans activating crRNA). This RNA had perfect complementarity to a region of the crRNA. Thus, Vogel and Charpentier believed that this RNA might have something to do with the functioning of the CRISPR system. They proved via knockout experiments, that this region was necessary for the proper functioning of the system. (3) fi ffi fi ffi ff fi fi fi 6 By this point, the necessary components of the CRISPR system - crRNA, tracrRNA and Cas9 - were known. It was time to see then how far this system could go. Making of the tool The rst step in application of the CRISPR system was to see whether a CRISPR locus from one organism could function normally in another organism. Siksnys and his group proved this by transferring the CRISPR locus from Streptococcus thermophilus to Escherichia coli and observing that the system functioned normally despite being in a di erent microbe. (16) Siksnys then studied whether the same function would be possible in vitro. He puri ed a streptavidin bound Cas9 and then showed that this Cas9 functioned the same in vitro. In the same experiment, it was also shown that spacers in the CRISPR locus can be manipulated in whichever way desired to target di erent sequences and the system would still function the same. (17) This occurred in 2012. For any of those who are familiar with the recent Nobel Prize (the year 2020) awarded for designing the CRISPR/Cas9 technology, I am sure they would know that it was in this very year that Jennifer Doudna and Emmanuelle Charpentier published the revolutionary article which launched CRISPR/Cas9 as a tool of genetic editing. What Doudna and Charpentier did di erently from the group of Siksnys was that they combined the crRNA and tracrRNA into a singular construct and called it the single guide RNA (sgRNA). They showed in vitro that this sgRNA alone was enough to allow Cas9 to perform its nuclease activity. In the very same article, Doudna and Charpentier commented that this tool had the potential to be exploited as a system for ‘RNA-programmable gene editing’. The article therefore was aptly named, “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity” (18) Figure-1 contains the timeline of the events covered in this section in brief along with some major discoveries in recent years. At the end of 2012, the journey from the initial discovery of the repeats to the construction of a functional tool was completed. It was time then to utilise this tool and see how far it could be pushed. Figure-1: A Timeline of CRISPR/Cas related discoveries ff ff fi fi ff Reprinted from Nidhi et al, Novel CRISPR–Cas Systems: An Updated Review of the Current Achievements, Applications, and Future Research Perspectives (103) 7 The Present Scenario: Basics of CRISPR/Cas9 system: Before we dive into the myriads of applications of the CRISPR/Cas9 system, it is important to possess a basic understanding of how it functions. It is better to understand the adaptive immune system as a whole rather than just the technique because the more knowledge one possesses, the more creative one can be. The functioning of the CRISPR/ Cas based adaptive immune system consists of the following steps1. Spacer acquisition 2. Biogenesis and maturation of CRISPR RNAs 3. Interference Spacer Acquisition: The CRISPR locus The rst step in the process is the capture of invading DNA and then adding it into the CRISPR locus so that a crRNA can be produced to allow the machinery to target the invader's genome. (19,20) The CRISPR locus is structured as follows - an AT-rich leader sequence is present right before the CRISPR repeats. As the leader sequence ends, you encounter the rst repeat which can range in size from 21-55bp, though usually, the size is somewhere between 21-47bp. (21,22,23) This repeat is followed by a spacer of almost the same length which can bind to the target, this is then immediately followed by another repeat and in this manner, the repeat-spacer-repeat pattern continues. (22) This region containing the leader sequences and repeats is anked by the CRISPR associated genes (position varies from organism to organism) which code for the expression of Cas proteins. (4) It is in this structure that a part of the invader’s genome must be added. The invader need not be a pathogen. (10) The process The process of addition of a new spacer is also called adaptation and the new spacer which is added is called the protospacer. (21) It begins with the recognition of foreign DNA. The di erence between host and foreign DNA (bacteriophage or plasmid) is chi sites. Spacer acquisition requires AddAB (gram positive bacteria) or RecBCD system (gram negative bacteria) to cleave regions of the invaders genome in order to insert it into the CRISPR locus. These chi sites reduce the activity of RecBCD and AddAB proteins. It must be intuitive then that these chi sites are highly enriched in the host genome as compared to the invader’s. There is a bias then of the machinery towards cutting DNA which has a lower amount of these sites. Another bias is produced by the presence of free DNA ends. The bacterial chromosome is circular in nature and thus contains no free ends of DNA, however, often, the genome of an invading bacteriophage is linear in nature, which allows the cutting machinery to home in on these sites. (24,25,26) The next step is to pick out a region of the invader’s genome which can serve as a protospacer. This selection is made on the basis of the presence of a 3 nucleotide fl fi ff fi 8 sequence called the protospacer adjacent motif (PAM). Genomic regions which are present immediately upstream of the PAM are picked up for use as a protospacer. The PAM is an absolute necessity for the system to perform both spacer acquisition and interference. The CRISPR locus of the host lacks this PAM and thus, there is no selftargeting. (19,21) The PAM recognising domain in Cas9 protein is responsible for detecting it and thus, it aids in protospacer acquisition by cutting the region present directly upstream of the PAM. The cleaved protospacer is then added into the CRISPR locus at the front end, directly downstream of the leader sequence and used for production of CRISPR RNAs. (19) This process can be di erent for di erent types of CRISPR/Cas systems, for example, type-I systems use Cas1-Cas2 complex to perform this function, as opposed to Cas9. (27,28) In type-I systems, spacer acquisition occurs at a lower rate when there are no pre-existing spacers present in the CRISPR locus which have either complete or partial homology to the invader’s DNA. In case a pre-existing spacer exists, the rate of spacer acquisition is much faster. This is called ‘Primed spacer acquisition’. (29,30) Biogenesis of CRISPR RNAs: The newly acquired spacer or pre-existent spacers are now transcribed to form the precrRNA. This long piece of RNA must be processed in order to convert it into a mature crRNA which can be used to guide Cas proteins to perform interference. For the purpose of maturation, exist proteins which di er from system to system. Cas6 protein is responsible for maturation in type-I and type-III CRISPR/Cas systems. The Cas6 protein cleaves the pre-crRNA in such a manner that a 5’ tag is present at the end of the process. Subsequently, an unknown protein causes further shortening of the RNA and leads to the production of a 3’ end which forms a secondary structure in the form of a loop. This completes the maturation process. (31,32) For type-II systems, the mechanism is entirely di erent which is what we shall cover here. In type-II Cas systems (Cas9 is the interference protein), a tracrRNA is required. This tracrRNA has a nucleotide sequence which is complementary to the repeat containing region of the pre-crRNA. TracrRNA binds to pre-crRNA, an interaction which is stabilised by Cas9. This tracrRNA:crRNA duplex is recognised by RNaseIII which cleaves this duplex and forms an intermediate crRNA. The nal step in the conversion of intermediate crRNA to mature crRNA remains unknown. (15) After a mature crRNA is produced, the last step of the process is interference. In order to keep the review brief, the interference mechanism of only CRISPR/Cas9 will be covered as anything beyond it does not qualify under the scope of this review. Interference: In this stage, the mature crRNA acts as a guide for the Cas proteins to speci cally target invader DNA in order to introduce double stranded breaks (DSBs) in it. (21) In CRISPR/Cas9 based interference, the tracrRNA:crRNA duplex is responsible for guiding the Cas9 protein to the target and introduce a DSB. Cas9 rst binds to the guide RNA via direct interactions with the secondary structures formed by the RNA. (33) This forms the structure necessary for search of target DNA. The detection of target DNA occurs in a stepwise manner where the Cas9 rst searches via assessing for presence of fi ff fi ff ff fi fi ff 9 a PAM. Upon nding it, base complementarity between the crRNA and the target DNA must occur for the recognition to be completed. In case the PAM sequence is not correct or if the crRNA and the region anking the PAM are not complementary, Cas9 rapidly dissociates away and no nicking takes place. However, if everything checks out, Cas9 dwells in that region for a long time and can conduct its endonuclease activity. (34) When an interaction between a PAM and Cas9 occurs, local DNA structure is altered via the introduction of a kink and turn, directly upstream of the PAM sequence. This kink and turn of the target DNA ips a nucleotide of the non-target strand thus making the target strand available for crRNA, hence facilitating interaction between the crRNA and the target DNA. Base complementarity then takes over and in this manner, a duplex is formed between the crRNA and a single strand of the target. (34) The displaced DNA strand of the target (also known as R loop) is stabilised by a large number of van der Waals and hydrophobic interactions. (35) The Cas9 is now ready to cleave the target DNA. Cleaving of target DNA is performed by two domains of the Cas9 protein, the RuvC and HNH nuclease domains. Each domain is responsible for cleaving one strand of the target DNA 3 base pairs upstream of the PAM. Such a nicking process creates a blunt ended DSB. (34) During the process of cleaving, the HNH and RuvC domains of Cas9 communicate with each other. After the R loop is formed, Cas9 undergoes a conformational change which brings the HNH domain closer to the target DNA. The conformational change in Cas9 causes crRNA strand invasion to move further. More base pairing causes greater conformational changes till an active conformation of the HNH domain is achieved. This active conformation in turn allosterically controls the RuvC domain. HNH is connected to RuvC through two linkers (namely L1 and L2) which allow for communication between them. These two linkers undergo large conformational changes themselves and direct the non-target strand towards the catalytic region of the RuvC domain. It is at this point that the Cas9 protein is completely active. The target strand is cleaved by the HNH domain and the non-target strand, after being inserted into the RuvC domain, is cleaved. Subsequent to the cleavage, Cas9 remains attached to its target until some other factors remove the enzyme in order to recycle it. (34,35) Figure-2: A schematic of CRISPR/Cas9 based interference The process of CRISPR/Cas9 based activity has 3 major steps. First, the crRNA is transcribed from the CRISPR locus (called biogenesis). Second, this newly transcribed RNA (called pre-crRNA) is cleaved in order to form a mature crRNA construct that can interact with tracrRNA. This process of cleaving of pre-crRNA is called maturation. The mature crRNA and a tracrRNA present inside a Cas9 protein interact to form the crRNA:tracrRNA duplex and thus the CRISPR/Cas9 construct is ready for target recognition. The nal step of the process is recognition of the target genomic region by the guide RNA. The target is recognised by nucleotide base complementarity between the crRNA and the target. After the target region is recognised in such a manner, it is then cleaved by the Cas9 enzyme. This process is called interference. (Nuances of the process have been mentioned in the main text of the review.) Schematic created with BioRender.com fl fl fi fi 10 Major variants of CRISPR/Cas9 system in use today: In this section of the review, we shall go through the creative ways in which this technique has been applied. CRISPR/Cas9 is exible enough that one is limited only by their own imagination. Variants of CRISPR/Cas9 come under two major categories: Variants in gene editing and variants which are used in functions other than gene editing. Cas9 enzyme variants in gene editing: As mentioned earlier, Cas9 enzymes assess a PAM in order to recognise their target and start the base complementarity and interference process. Then, do all Cas9 enzymes have the same PAM preference? The answer would be no. The fun of biology lies in the inherent diversity and variations in systems across a wide range of organisms. As such, there are Cas9 enzymes which have di erent PAM preferences. Not just that, Cas9 enzymes vary in their sizes and sgRNA requirements as well. (36,37) This creates for us a large pool to sift through in order to nd a Cas9 enzyme which suits our requirements the most. Let us look at these Cas9 enzymes rst. The various Cas9 enzymes in use today have been mentioned in table-1. Such a large variety of Cas9 enzymes is in use today due to various reasons ranging from compatibility to target organism (mostly PAM compatibility), the size of the Cas9 enzyme, size of the guide RNA etc. (36) Factoring these variables, the Cas9 currently in rst place as far as usage goes is SpCas9 (Streptococcus pyogenes). The obvious reason behind it is the elementary PAM requirement - NGG - where N could be any nucleotide. This allows for more target exibility as compared to other enzymes. fnCas9 does possess the same PAM requirement, but the fact that it is larger in size as compared to spCas9 limits its usage. Size matters because the CRISPR assembly needs to be delivered into the target organism and a larger size means more problems in e cient transfer of the CRISPR/Cas system into the target organisms. Although there are Cas9 enzymes which are smaller than spCas9, it turns out that natural Cas9 enzymes which are smaller in size have more complex PAM requirements, thus the game becomes a balancing act. A larger size means more di culty in delivering the assembly, a smaller size means more PAM complexity. One thus has to balance these trade-o s in order to pick the enzyme most suited for their use. It is due to the aforementioned reasons then that spCas9 dominates the eld (36) Cpf1 enzymes have been mentioned in the table as well since they allow for much more e cient target cutting, are smaller in size as compared to most Cas9 proteins, require only one guide RNA as compared to a tracrRNA:crRNA duplex, are responsible for producing mature crRNA on their own and do not rely on any other enzyme for this function. Beyond all that, they cut downstream of the PAM sequence and introduce staggered cuts as compared to upstream and blunt ended cuts, which is what Cas9 fi ffi fi fi ff ff fi fi fl ffi 11 ffi fl Figure-2 consists of a schematic which shows the major steps of the entire process. Many nuances have been skipped in order to make the mechanism easier to understand and to stay within the scope of the review. To understand more about the mechanism, it is recommended that the reader follow up on the speci c references mentioned in this section. With a basic understanding of how CRISPR/Cas9 works, we shall now turn the discussion towards the applications of this revolutionary technology. enzymes do. This makes them much more reliable for genetic editing as compared to Cas9, yet these enzymes still have not overtaken Cas9 enzymes as far as mainstream use is concerned. Some reasons for this are that the Cpf1 enzymes require tighter temperature control in order to function properly and because the size of the guide RNA is smaller, there are greater chances for secondary structure formation which may be di erent from what is desired. Besides that, Cpf1 enzymes can be used for any function that Cas9 enzymes can perform. Please keep that in mind as we explore the various applications of the CRISPR/Cas9 system. (37,38,39,40) This table lists the details of all the major Cas9 enzymes currently in use today. Two Cpf1 (now Cas12a) enzymes have been mentioned as well since their impact cannot be entirely ignored. (36,37) Terminology used N = Any nucleotide; R = Pyrimidines; W = Adenine or Thymine; V = Guanine or Cytosine or Adenine. The idea behind Cas9 based gene editing - ff Cas9 via the use of a sgRNA can be used to target any region of the DNA. By now, it must be clear that this statement is not completely accurate. Using Cas9 and a sgRNA, you can target regions of the DNA which have the appropriate PAM following it or preceding it (depending on whether you are using Cas9 or Cpf1). The manner in which Cas9 based genome editing works is quite simple. The scientist relies on the ability of the tool to create a DSB. Once this double strand break is created, it is detected by an organism’s 12 repair machinery. At this point, NHEJ (non homologous end joining) or HDR (Homologydirected repair) takes over. (20) Each of these mechanisms has di erent usages. In case a scientist wants to produce a knockout, NHEJ is preferred. NHEJ despite being a method of DSB repair for DNA, is prone to either cause some deletions or random insertions. Introduction of either of these leads to an alteration in the genetic code of DNA (based on codon triplets). Due to the introduction of or removal of nucleotide bases, a frameshift occurs. This shift causes alterations in the way the mRNA produced by this DNA fragment is read for translation. In such a manner, a non-functional protein is produced. Thus, a gene is e ectively knocked out. HDR is employed when you want to perform a targeted gene deletion, or to insert a gene or a gene segment into the genome. This has been described in Figure-3. (20,41) How to use the tool The following considerations must be made in order to use the tool: 1. Choice of target DNA- It is obvious that your target of choice should always be next to a PAM, otherwise your experiment will not work. A useful tip here is to target a region as early on in the coding region of the gene as possible. Doing this will accentuate the e ect of deletions or insertions caused by the system. There will still be a lot of trial and error as the design of your sgRNA will depend on not only the target, but also the organism in which you are performing the experiment. (42,43) 2. sgRNA design - An sgRNA must contain the following 3 regions, a guide sequence which is complementary to the target DNA, an intermediate region which resembles the formation of a duplex between crRNA and tracrRNA and nally, it must contain a tracrRNA region which folds to form secondary structures in a manner such that a Cas9 enzyme can bind to the sgRNA. sgRNAs must be designed to target regions close to the preferred PAM for your Cas9 enzyme. The promoter to be used to express your sgRNA must be appropriate for your target organism (as in, whether your system is a prokaryote or a eukaryote). It is also pertinent to mention here that the GC content of your sgRNA should not be too low or too high. Further, repeated nucleotides at a stretch have been shown to reduce the e ectiveness of the sgRNA. In order to support you, there are multiple sgRNA design tools available online, including one from Zhang lab. You can employ these to design a guide RNA as per your speci cations. (20,44) 3. Delivery method - The nal step in the process is picking out what you will use to deliver the CRISPR assembly inside your organism. Methods of delivery include simple methods such as plasmid vectors which have the CRISPR assembly under the action of inducible promoters in order to control the function of CRISPR/Cas system. For experiments which go on for a while, it is better to knock in the CRISPR assembly into the chromosome of the organism and create a stable cell line such that your CRISPR locus is faithfully replicated in each generation. Such methods are employable for gene knockout or knock in experiments. But, in case of gene therapy, Adenovirus based systems are employed but this comes with a lot of complications (for example immune responses generated by the adenovirus) and are beyond the scope of this review. (45,46,47) Now that we know how to apply the tool (at a basic level), it is time to start exploring its applications in the scienti c world. ff fi ff ff fi fi ff fi 13 Applications of CRISPR/Cas9 in genome editing Because of its ease of use, this tool has been used extensively in reverse genetic studies to understand the functions of genes (gene knockout experiments), understanding diseases and in gene therapy as well. By using targeting via sgRNAs properly, genes can be knocked out and corresponding changes in phenotypes can be studied. (36) Using sgRNAs for targeted knock in for genes, one can produce transgenic organisms in order to develop model organisms for study of diseases. This has been achieved in fungi (48), plants (49) and a large number of animals as well. (50) CRISPR/Cas9 excels more than other tools at such feats simply because of its ease of use. Applying this concept at a wider range, a large number of sgRNAs have been used in a system at the same time to achieve large scale alterations. An instance would introducing 2 DSBs in the same segment of DNA so as to cause an inversion of the gene segment. The same method can be used to cause translocations by producing DSBs in multiple segments. All this may sound speculative, but these methods have been used in experiments already. Such chromosomal alterations have been employed to study oncogenesis (51), disease progression (52) and e ects of chromosomal translocations. (53) The same method can then be applied to perform large scale screens to understand every aspect behind any given biological event. This technique has also been used for gene therapy of genetic disorders such as cystic brosis, and the infamous case of He Jiankui, a scientist who edited genomes of human babies in order to make them less susceptible to HIV. These applications are beyond the scope of this review, but are interesting reads. It is encouraged therefore, that you go and read about these events. (20,54) Figure-3: A schematic depicting CRISPR/Cas9 based gene editing CRISPR/Cas9 based gene editing relies on the repair mechanisms inside organisms to produce its effects. NHEJ is used to mainly disrupt or knock out your gene of interest as this method of repair is error prone. HDR is used when you want to knock in a gene, perform a targeted deletion or correct a gene of interest. fi ff Schematic created with BioRender.com 14 CRISPR/Cas9 beyond gene editing: It is beyond gene editing where CRISPR/Cas9 has left all other genome editing techniques in the dust. We shall cover these applications one by one now. Most of these techniques employ a catalytically dead cas9 enzyme (dCas9) which has been arti cially engineered to remove its nuclease activity. This is achieved by introducing targeted mutations in the HNH and RuvC domains of Cas9 enzyme in order to prevent cleaving of target DNA. (36) Section by section, we will now explore the application of the Cas9 enzyme beyond prototypical gene editing. Control of gene expression dCas9 has been used beyond gene editing in creative ways. A dCas9 enzyme possesses the ability to bind to target DNA but does not cleave it. This property has been employed to cause interference in the genome to cause gene repression. This technique is named CRISPRi for CRISPR interference. The concept behind it is that upon target recognition, dCas9 binds to the target region. This binding prevents other enzymes from accessing this region of the genome which leads to its silencing. The advantage of this technique is that we can mix the inducible promoter trick mentioned earlier along with CRISPRi to temporally control the silencing of a gene and when required, we can revert it to make the gene functional. Such an application is important when studying genes which are essential for cell development or growth. Thus, CRISPRi knocks down a gene instead of knocking it out. Besides this, one can attach a repressor to a dCas9 and then target this repressor to the gene of interest to silence it. An example of such a repressor are KRAB domain containing proteins (Krüppel associated box containing protein). The addition of a repressor enhances the silencing ability of dCas9 and allows for more accurate targeting. (45,55) Opposite to this, you can attach an activator to the dCas9 in order to enhance expression of a protein. This methodology is called CRISPRa (CRISPR activation). To achieve this, you can introduce a dedicated activator of a gene and couple it to dCas9. This will recruit proteins to the target site thus inducing your gene of interest. Nowadays, this technique has evolved to an unprecedented level. Instead of a single activator, scientists have used activator complexes in order to achieve a higher level of induction. To provide an example, Chavez et al. used a tripartite complex. In their article, VP64 was used as an initial activator and a corresponding increase in transcriptional activity was noted. Following that, they created bipartite fusions with the C-terminal domain of the dCas9VP64 complex, using either p65 or Rta. These double activator complexes were more e cient in inducing the gene of interest as compared to the individual activator. The nal step was to create a tripartite complex where both p65 and Rta were fused with the dCas9-VP64 complex. This complex achieved the highest level of activation! (56) In this manner, one can manipulate gene expression tightly using dCas9 according to their will. It is possible to simultaneously introduce orthogonal activator and repressor constructs attached to dCas9s under di erent inducible promoters to control expression of a gene of interest and study its function. fi ff fi ffi 15 Shining a spotlight on chromosomes, live cell imaging with Cas9 In this variation, instead of attaching an activator or repressor to the dCas9, one can simply attach a uorescent protein to it and target any genetic locus of interest. By doing this, you can view genomic structures live and understand their organisation. dCas9 has not exactly revolutionised this methodology. TALEs (transcription activator-like e ectors), that are another type of DNA targeting proteins, have been used before to perform similar experiments, however, dCas9 being more exible, has overtaken other methods of live cell imaging. Such a technique is a far cry from the days of uorescence in situ hybridisation where a cell would have to be heated and xed before making observations. Thus, a considerable jump has been achieved in live cell imaging using CRISPR/Cas9. (57,58,59) Alter chromatin architecture at your whim Large scale chromatin structure alterations can also be achieved using dCas9 tethered with appropriate proteins. A study by Morgan et al. used dCas9 enzymes tethered to ABI1 or PYL1. They introduced plasmids with the dCas9-ABI1/PYL1 constructs in human bone marrow cells and achieved large scale chromatin structure manipulation. The idea behind the technique is that 2 di erent dCas9 enzymes are tethered to two di erent proteins, ABI1 and PYL1. The trick is that these two proteins, which are bound to independent dCas9s have the ability to bind to each other. So, when two of these constructs bind to two di erent target DNA regions, the a nity between the proteins tethered to dCas9s will cause them to bind, thus bringing target regions close to each other and altering the structure of chromatin. In the same article, they also showed that simply altering the chromatin structure is enough to induce expression of genes. They named the technique ‘reversible chromatin loop reorganisation using nuclease-de cient Cas9’ or CLOuD9 in short. (60) Controlling the epigenome Human DNA is packed tightly in order to t a large amount of genetic information into minuscule spaces. This packing is performed by the presence of proteins called Histones. DNA winds around histones and thus gets packed in tightly wound structures called nucleosomes. Consequently, genes which are packed tightly are silenced since proteins cannot reach them. In contrast, genes which are active are loosely packed to allow various enzymes access to those regions in order to transcribe and express them. The wondrous thing about nucleosomes is that by manipulating the modi cations of histones, the tightness of packing of a certain region of the genome can be controlled. Dedicated enzymes exist for these functions. Beyond that, DNA itself can be directly subject to modi cations (such as CpG island methylations). (61) You must get it by now. Dedicated enzymes for a given function? Existence of dCas9? Flexible targeting using sgRNAs? This combination of factors thus allows us to tether various epigenetic modi er enzymes such as Histone methylases, Histone acetylases, Histone deacetylases etc. to dCas9 in order to manipulate the targeted epigenetic landscape and thus, study the e ects of di erent epigenetic modi cations in a genetic region. Today, many such epigenetic proteins have been tethered to dCas9 to study di erent functions. (62,63,64) ff ff fi fi fl fi fi ff fl fi ffi ff ff fi fl ff fi ff 16 Split Cas9 A split Cas9 is an enzyme which has been literally split into two pieces. When split, the Cas9 enzyme will be inactive. In order to activate it, a substrate is added which allows the split Cas9 parts to interact and thus form a functional Cas9. At a deeper level, what needs to be done is to attach dimerisation domains to pieces of the Cas9 enzyme. These dimerisation domains can be substrate inducible like the one produced by Zetsche et al. (65) who used rapamycin binding domains tethered to split Cas9s. Introduction of rapamycin in the system activates the Cas9 by bringing the pieces of Cas9 together. Nihongaki et al. instead of using substrate based domains, used photo inducible domains called magnets which interact with each other only upon irradiation by blue light. (66) In this manner, by keeping the Cas9 separated and bringing it together with a special stimulus, greater spatiotemporal control of the system can be achieved. It is important to mention here, for any of the techniques mentioned above, inducible promoters which control the expression of Cas9 itself can be applied. By doing that, we can introduce layers of control in our experiment. As a result, accuracy of the system can be increased and o -target e ects of the same can be reduced dramatically! All of the strategies mentioned above have been summarised in gure-4. With this, we conclude our foray into the major variations of CRISPR/Cas9 techniques presently in use. In the next section, we shall explore the future of this technique. Figure-4: Various strategies of CRISPR/Cas9 application in use today The gure has been divided into two parts. The two strategies on the left are DNA editing strategies. (Direct targeted gene editing has been covered in the Basics of CRISPR section and Base editing, which has been explained in the future of CRISPR section.) On the right are strategies which employ the use of a dCas9 enzyme and thus are the applications which make use of the fact that the dCas9 enzyme can be targeted to a speci c genomic region using a sgRNA. Various effector enzymes or molecules are attached to such a dCas9 enzyme and thus, they receive a ride via dCas9 to the genomic region of interest. All of these strategies have been discussed in the CRISPR/Cas9 beyond gene editing section. Please refer to each section to learn more about these strategies. 17 fi fi ff ff fi Reprinted from Adli, M. (2018). The CRISPR tool kit for genome editing and beyond. In Nature Communications (Vol. 9, Issue 1). Nature Publishing Group Beyond: The major variations and techniques under the domain of CRISPR/Cas9 have been discussed. In this section, we move beyond the present and try to understand what lies in the future for CRISPR/Cas9. We shall look into the latest developments in the eld and understand the obstacles that are being tackled in order to evolve the technique further. Finally, we shall cover the human side of science, the ethics of the application of such a technique. Future of CRISPR/Cas9: Second generation CRISPR tools: Second generation CRISPR tools signify a leap from random base alterations to targeted base alterations. These tools tether a di erent type of enzyme to the dCas9 protein so that bases can be edited. The dCas9 tethering to an enzyme trick is not exactly a new idea, but by using an entirely di erent class of enzymes, one can alter bases in a genome without introducing DSBs. Imagine the exibility this would provide! The routine Cas9 simply causes DSBs and it is the NHEJ repair machinery which then causes deletions or insertions. Of course, this would be non-speci c. HDR could be an option then? But, it turns out that even when HDR is supposed to happen, the cell can choose NHEJ based repair because it is more e cient, leading to undesired results. But in the case of Second generation CRISPR tools, without breaking the DNA at all, one possesses the ability to edit DNA by making speci c and targeted base alterations. (36,67,68) This shall be explained further. Liu et al. create the rst CRISPR/Cas9 base editor In 2016, from the laboratory of Liu came out an article which described a paradigm shift in the world of CRISPR/Cas9. What prompted their research was the information that upon binding of Cas9 to the protospacer in the target DNA, an R loop is formed i.e. ssDNA (single stranded DNA) is present in that location. Cytosine bases of a segment of ssDNA which is exposed are prone to deamination by enzymes called cytidine deaminases. Removal of an amino group by these enzymes converts a Cytosine base into Uracil. This Uracil during the course of replication is then read as a Thymine. Thus, a deamination event converts a C-G pair into a T-A pair. In their study, di erent cytidine deaminases were tethered with dCas9. The highest e ciency was achieved by the rat APOBEC1. The enzyme in vitro and in vivo (albeit at a lower e ciency) converted Cytosines to Uracils which were replicated in progenies as a T-A pair. This was the rst generation of Base editors (BE1) with lower in vivo e ciency rates because of the cellular repair machinery which could detect the U-G mismatch pair formed after the deamination and correct it. To avoid the cellular repair, an enzyme called Uracil DNA glycosylase inhibitor was fused to the C terminal end of the BE1 complex. This construct was called BE2. What this BE2 could accomplish was that it could inhibit the enzyme responsible for the repair of the U-G mismatch. A higher e ciency was achieved in vivo with this base editor. Liu and colleagues were still not satis ed however. The nal step in the evolution fi fi ff fi ffi ffi ffi fi fi ffi ff fl ff fi ffi fi 18 process was to restore some of the cleaving activity of dCas9 by reverting the mutations in the HNH domain of dCas9 to form an enzyme called the nickase Cas9, which would cleave only one strand of DNA instead of both. What this nickase Cas9 could do was to bias the cellular repair machinery to use U as the template for repair of the U-G mismatch pair instead of G. Thus, prompting the repair machinery itself to create the edit by nicking the strand which contains the G. So, the outcome of this kind of repair would be a U-A pair which would then become a T-A pair during replication. This was the rst instance of targeted base editing using CRISPR/Cas9 without the introduction of DSBs. (69) In the years since targeted base editing was achieved, a lot of modi cations have been made to the base editors. An example would be Koblan et al. attaching nuclear localisation signals to the base editor complex in order to improve nuclear localisation of BEs and thus increase their e ciency. This led to enzymes called BE4max and ancBE4max. (70) More cytidine deaminases have been found since then, with di erent sequence preferences in order to widen the pool of targets which can be altered. (68) Many developments and improvements have been made since then, a good amount coming from Liu’s group. It is encouraged then that the reader follow his work if they want to explore base editing further. C-G to T-A isn’t all of it Cytidine deaminase developed by Liu and colleagues allowed for C-G to T-A base pair transitions. After they achieved that in 2016, they did not rest on their laurels. In 2017, they published another article, describing an Adenine deaminase which caused an Adenine to Inosine conversion, the inosine then being read as a G, converted an A-T pair into a G-C pair. The process seems simple, but developing an enzyme which converts AT to G-C was not easy. It turns out, Adenine deaminases don’t occur naturally. Even if they do, they only perform deamination of Adenosine bases present in RNA, not DNA. The di culty then was to engineer an enzyme which causes deamination of Adenosine in DNA. To achieve that, they took an adenine deaminase from Escherichia coli (ecTadA) and tried to evolve it into an enzyme which would be able to process Adenine using DNA as a substrate. The trick they used was ingenious. They tethered this ecTadA to a dCas9 and in the genome of the organism, introduced faulty antimicrobial resistance genes which would become e ective only if there were to be a base pair conversion from A-T to G-C. They grew these organisms with the ecTadA-dCas9 fusion in the presence of the antimicrobial and selected for a mutant which could survive. In this manner, they got an adenine deaminase which could target DNA. (71) Since then, much more e cient and e ective adenine deaminases have been developed. Scientists have used techniques of evolution such as Phage assisted continuous evolution (PACE) or Phage assisted non-continuous evolution (PANCE) to enhance these enzymes. These enzymes show greater catalytic activity and greater compatibility to a larger variety of dCas9s, hence improving their exibility of use. (72) So, with these classes of enzymes, you can achieve four DNA base transitions, making precise DNA base editing applicable in a large amount of instances. Going beyond four transition mutations - ff fi fi fl ff ffi ffi ff ffi Anzalone et al. recently described a method of editing bases precisely using a reverse transcriptase attached to a nickase Cas9 and an important piece, the prime-editing guide 19 RNA (pegRNA). The prime editing guide RNA has a twofold function. It guides the nickase Cas9-reverse transcriptase assembly to the target site and it also serves as a template for the reverse transcriptase to generate DNA from the pegRNA. The newly synthesised DNA can then bind to the target via base complementarity and then get faithfully reproduced during replication. A caveat in this process is that two complimentary strands exist at the end of this process, one strand that was bound to the target originally and another strand which is synthesised by the reverse transcriptase. It turns out that the original strand that is selectively degraded by cellular endonuclease as compared to the newly synthesised strand which causes the equilibrium to shift towards the newly synthesised DNA strand. In this manner, using the guide RNA itself as a template, base edits were made. Third generation prime editors exist today which are much more e cient and thermostable as compared to their ancestors. (73,74) This method of editing bases has been termed ‘Prime editing’ and allows one complete freedom to edit bases in whichever way they seem t. Of course, the normal limitations exist for this system as well, some of which are PAM speci city of Cas9, problems with the delivery of the system and a ected e ciency of the conversions due to cellular repair machinery. Greater e ciencies have been achieved however for Cytosine BEs, Adenosine BEs and Prime BEs by using secondary sgRNAs to nick the non-template strand such that the cellular repair machinery uses the edited DNA strand as a template rather than the non-edited one. This would shift the equilibrium towards the desired outcome. (68) Precise base editing has allowed applications such as generating premature stop codons (for example converting a TGA into a TAA) in order to knockout genes (CRISPR STOP). (75) One could also tackle diseases which are caused due to a SNP (single nucleotide polymorphism) and use editing to permanently cure the disease. Despite their recent advent, precise base editors have been able to work their way into mainstream CRISPR/ Cas9 applications. (68) Much work still needs to be done to avoid o target e ects of these editors and to increase their e ciency. It is only a matter of time then that these methodologies start dominating the eld of therapeutic genetics. Pushing CRISPR/Cas9 even further: In this section, it is basically a free for all. It will contain descriptions of the latest techniques and instances where some of the major obstacles to achieving the perfect CRISPR/Cas9 construct have been tackled. Thus, this section is basically a treat for any fans of CRISPR/Cas9 (courtesy of the author). Controlling CRISPR/Cas9 Throughout this review, mentions have been made regarding o target e ects of the technique. It is important to be able to control the assembly tightly so as to avoid these e ects. We have gone through inducible promoters for Cas9 and split Cas9s which themselves can be under the control of di erent inducible substrates, but even then, after the machinery is activated, it stays in the cell for some amount of time which could lead to o target e ects. So, to counter that, we shall discuss 2 solutions, sgRNA based control and Anti-CRISPRs. ff ff ff ff ffi fi fi ff ffi fi ffi ff ffi ff ff ff 20 sgRNA based control In 2020, Hao et al. described a method of controlling the CRISPR/Cas9 system by focusing on the sgRNA rather than the Cas9 itself. They described oligonucleotide based constructs with a toehold at the end which would allow precise control of the CRISPR/ Cas9 assembly using oligonucleotide inputs. (76) Cas9 needs to bind to the tracrRNA region of the sgRNA in order to be guided to the target. This binding occurs due to the presence of secondary structures in the tracrRNA region. What Hao and colleagues did was that they designed oligonucleotides that were complimentary to the secondary structures of the sgRNA such that base complementarity between the oligonucleotides (blockers) and the sgRNA regions would cause these blockers to bind to the sgRNA, a ecting the secondary structure of the sgRNA which is necessary for its interaction with Cas9. As a result, these blockers strip the ability of the sgRNA to bind to Cas9. Another ingenious trick they used was at the end of these oligonucleotides, they added bases which were not complimentary to the sgRNA. These bases would then hang away from the oligonucleotide-sgRNA complex and allow for another oligonucleotide (input) to bind to these bases. The technique works as follows. Initially, the sgRNA and blocker are co-expressed. The sgRNA and blocker bind and thus Cas9 is unable to bind to the sgRNA (OFF-state). Now, when the assembly needs to be activated, you arti cially insert inputs which would be complimentary to the hanging region of the blocker. This would cause the blocker and input to bind to each other thus freeing the sgRNA to form its secondary structures. The free sgRNA would then interact with Cas9 and guide it to the target (ON-state). Now, for a little while the assembly will be active, but after a certain amount of time, the equilibrium between the blockers and inputs will shift since blockers are generated internally as compared to the arti cially inserted inputs. In this manner, you would block the sgRNA again and the assembly would again be in the OFF-state. The same process has been described in Figure-5. (76) Figure-5: Modifying the sgRNA to control the CRISPR/Cas9 system a. A description of how base complementarity is applied to affect the secondary structure of the sgRNA and hence prevent it from binding to a Cas9 enzyme. b. Insertion of an input which is complementary to the toehold switch removes the blocking strand and thus, turns on the sgRNA. c. A schematic of the technique. A sgRNA is in the OFF-state when blocked using a blocking strand and upon insertion of the input, the blocking strand is removed and thus, Cas9 can interact with the sgRNA in its ON-state and produce a downstream effect fi ff fi Reprinted from Hao, Y., Li, J., Li, Q., Zhang, L., Shi, J., Zhang, X., Aldalbahi, A., Wang, L., Fan, C., & Wang, F. (2020). Programmable Live-Cell CRISPR Imaging with Toehold-Switch-Mediated Strand Displacement. Angewandte Chemie - International Edition 21 Thus, using strand displacement reactions, you can dictate when the assembly is on or o . Further, you can use di erent combinations of blockers and inputs such that only a speci c combination of inputs gives you the desired result. Hao et al. also go on to mention that using this methodology, you can construct logical gates for CRISPR/Cas9 function inside the cell. (76) Anti-CRISPRs Anti-CRISPRs (Acr) are another method of controlling CRISPR/Cas9. CRISPR exists as a defense against mobile genetic elements (MGEs), but since this is biology we are talking about, the recipients of the attacks by CRISPR/Cas will not just stay silent and take it. They will have to evolve in some manner. The answer various MGEs came up with was a group of proteins called Acr proteins. Acrs are a diverse set of proteins coded by MGEs which directly interact with CRISPR proteins and inhibit their functions. The stage at which inhibition occurs can vary from DNA binding by crRNA to cleavage of the DNA by Cas proteins. (77) Acr proteins were discovered by Joseph Bondy-Denomy by analysing phages that were escaping the wrath of the CRISPR/Cas system. After such a discovery, a search began, looking for such genes that could protect a phage from the CRISPR/Cas system. During that search, it was found that Acr proteins cluster together in the form of a loci, so despite having minimal similarities to each other or to Cas proteins, multiple Acr proteins were discovered and analysed. Since then, these proteins have found applications in regulating the CRISPR/Cas system. (78,79,80) In a system with a CRISPR/Cas construct, Acr proteins can either be introduced exogenously or can be introduced as a genetic construct under the control of an inducible promoter to be expressed whenever required so as to stop the function of the CRISPR/ Cas system. Thus, Acr proteins can be used to gain another layer of control over the CRISPR/Cas system in order to avoid the o -target e ects. (77) The most exciting possible application of this discovery would be in creating CRISPR/Cas based gene drives. Such gene drives would basically consist of releasing CRISPR/Cas engineered organisms into the environment at a population level such that the engineered locus spreads in a population via natural methods of reproduction. The engineered locus would then, after a certain amount of time, be present in all members of a population as they move through generations. An example of such a gene drive would be one designed by Gantz et al. who created a transgenic Anopheles stephensi (vector for malaria) so that it became resistant to the malarial parasite Plasmodium, making it impossible for the vector to carry the pathogen, hence stopping the spread of malaria entirely. (81) This would be the ideal scenario, however, questions have been raised against releasing organisms with modi ed genomes in the environment as it may have unpredictable deleterious e ects. However, imagine a scenario where you have an Acr system present in the gene drive alongside the CRISPR/Cas system. So, you always have a bona de ‘KILL SWITCH’ to stop the gene drive in case something goes awry. Thus, Acr proteins give rise to exciting new possibilities in the world of CRISPR/Cas. Beyond this, Acr proteins could also be used to reduce the pathogenicity of virulent organisms which depend on their CRISPR/Cas system for their defense. (77,81) fi ff ff ff fi ff fi ff 22 A bonanza of interesting leaps, techniques and applications Cas9 as a highly speci c biosensor Cas9 until now has been applied in genetic applications such as editing, silencing, even activating gene segments and other interesting applications such as targeted live cell imaging. Extending the live cell imaging technique a step further, what Guk et al. did was they used targeted CRISPR/Cas9 to detect a Methicillin-resistant Staphylococcus aureus (MRSA) using a fusion of dCas9 with SYBR green dye to target the mega region of the MRSA genome. Not only that, they were able to discern between a MRSA and Methicillin sensitive Staphylococcus aureus (MSSA). What they achieved was a a combination of Fluorescence in situ hybridisation and CRISPR to create a diagnostic construct, hence creating a CRISPR/Cas9 based biosensor for detecting MRSA. CRISPR/Cas9 in similar manners has been used to detect multiple bacterial resistance genes and to discern hostpathogen interactions as well. Since then, not only Cas9, but also other Cas proteins such as Cas12a and Cas13 have been used to design highly speci c diagnostic tools. (82,83) Looking for a PAM free Cas9 Many a time in this review, it has been mentioned that Cas9 requires a PAM in order to perform its function. This limits the diversity of gene sequences that are truly editable. In order to increase the pool of sequences that can be edited, scientists have been looking for naturally occurring Cas9s with di ering PAM preferences, hoping to nd that one hidden gem, which would be a Cas9 enzyme that does not require a PAM at all. Though that approach is ne, some scientists have decided to take a di erent route, arti cially engineering enzymes to produce di ering PAM speci cities. To that e ort, a large amount of engineered Cas9s have been developed. (84) An engineered Cas9 which is of great interest to the scienti c world was developed by Nishimashu et al. They designed a variant of SpCas9 called spCas9-NG. A wild type SpCas9 has a NGG PAM preference but after the introduction of mutation in the Arginine residue present at amino acid position 1335 of the SpCas9 and a follow up evolution experiment, a Cas9 was generated which did not need the second G of the PAM for target recognition. Thus, they developed a Cas9 which only required NG as its PAM. This signi ed a quantum leap in the kind of sequences you could edit. (85) Following this experiment, Walton et al. looked to truly generate a Cas9 without any PAM preference at all. They performed targeted mutagenesis (which they called structure guided mutagenesis) and generated two Cas9 variants. One was a SpG variant which recognised an NG sequence as PAM similar to Nishimashu’s group, but the second variant was SpRY. SpRY was a Cas9 enzyme which recognised the sequence NR as a PAM (where R = A or G). It also recognised a RY sequence (where Y = C or T) as a PAM, although to a lesser extent. As you can appreciate, having an enzyme which can recognise an NA or an NG sequence as a PAM would dramatically increase the variety of genetic sequences you can target. Thus, this experiment was a great leap in the world of engineered Cas9 enzymes. To this day, SpRY is the most exible Cas9 enzyme. (86) Since then, many scienti c groups have been looking to create a PAM free Cas9. In the pool of such literature, an article has been gaining some steam recently. This article was written by Qi et al., the contents of which describe creating a SpRY toolbox which allows fi fi ff ff fi fi fl fi ff ff fi fi fi fi 23 them to nearly edit the entire plant genome at their will. The word PAM-less was mentioned in the title of their article and in a Scitech interview, they mentioned that this toolbox removes the biggest bottleneck as far as CRISPR/Cas9 genome editing is concerned, which is the PAM. (87,88) What this article signi es is a paradigm shift in the search for a PAM free Cas9. It introduces a new approach. The approach of combining di erent Cas9 enzymes so as to have a repertoire that together can target all possible PAM combinations allowing us to e ectively edit any DNA sequence, rather than searching for that one magical enzyme which can edit all sequences. This approach is being adopted by the scienti c community as there is increasing literature which talks about lling the holes in the current PAM repertoire as compared to just searching for one PAM free nuclease. Other reasons also can be attributed to this shift away from a PAM free nuclease. A truly PAM free Cas9 would have greater o target e ects and would demand more stringent control as compared to a Cas9 which has a preferred PAM. Even beyond that, a PAM free Cas9 would have to assess the entire genome for its target and as a result, the time taken for such experiments would increase by a large amount. These trade-o s will have to be considered if one day we truly achieve a PAM free Cas9. But, based on the current trend, a complete Cas9 repertoire is the direction which seems more appropriate. (84) Remote controlled gene editing? We have read about light inducible split Cas9s, however, what those Cas9s require are tedious systems such as a laser or an optical bre in order to activate those Cas9 enzymes which limits the exibility of the technique. Yu et al. designed a system to remove that barrier. They developed a system called FAST which stands for Far-red light (FRL) activated Split Cas9 system. This system allowed them to use LED lights to shed far red light on mice and achieve photo inducible editing in the internal organs. (89) The FAST system was constructed using two FRL sensors - BphS and p65-VP64-BldD; and two cohesion proteins - DocS and Coh2. They fused these to a split dCas9 enzyme and thus produced a system which upon induction by Far-red light would fuse the split Cas9s together and allow them to perform their activity. This system pushes the optical repertoire for Cas9 which usually would require phototoxic wavelengths of light in order to be activated. Using just an LED for the same function would allow photoinducible Cas9 control in vivo without damaging the organism. (89) Knock in anything! CRISPR and Transposons join hands Knocking out using CRISPR is fairly easy. Knocking in is much more di cult. Especially with large genomic segments, the e ciency of the CRISPR/Cas9 system plummets because it depends on HDR. In contrast to Cas9, transposons have the ability to insert large genomic segments, however, they lag behind CRISPR/Cas9 in its ability to target speci c targets. So, it was easy for someone to imagine a scenario where Cas9 could take care of the guiding function and transposons could take care of the knocking in function. Came forth a fusion of the two, CasTn system. Cas-Tn means Cas-Transposon where a transposase (Himar1) is fused to a dCas9 enzyme to create a targeted transposase construct. Using this assembly, Sway Chen and Harris Wang were able to achieve a targeted knock in at a rate which was greater than 300 times than that of an isolated transposase on its own. In the study, by using knockdown of uorescent proteins such as mCherry and GFP, they showed that their construct could achieve targeted knockdowns. Further, using a promoter less puromycin resistance gene as a payload for ffi fl ff fi ff fi ff ffi fi fl ff fi fi ff 24 delivery, they displayed that the CasTn could be used for targeted insertion of genes. They therefore demonstrated that Transposition and CRISPR/Cas9 can be combined to create a novel and much more accessible platform for gene knockins as compared to what these two systems could achieve by themselves. The process has been explained in Figure-6. (90) Figure-6: A schematic depicting the process of targeted knock in of a transposon via tethering to a dCas9 enzyme A Himar transposase is tethered to a dCas9 enzyme along with the transposon which is supposed to be knocked in. This assembly is guided to the region of interest using the sgRNA which nds the target genomic regions and binds to the dCas9. Thus, the entire assembly is driven to the region of interest where via the action of the transposase, the transposon is knocked in. Reprinted from Chen, S. P., & Wang, H. H. (2019). An Engineered Cas-Transposon System for Programmable and Site-Directed DNA Transpositions. The CRISPR Journal, 2(6), 376–394. With this ends the fan service. All of these techniques and applications serve as a possible direction of CRISPR/Cas9 development. Thus, the future itself is the future perspective. As it is impossible to entirely predict human behaviour, it is also impossible to predict the extent of human creativity. Thus, these techniques here have been mentioned to serve as some ideas that might allow you to maybe one day create a CRISPR/Cas9 variant of your own. As a follow up, there are many more interesting articles related to creative applications of CRISPR/Cas9 coming out everyday. The go to journal for accessing or keeping track of these articles would be the CRISPR journal which is a dedicated journal that keeps track of CRISPR based technologies. It can be accessed using this link: https://home.liebertpub.com/publications/the-crispr-journal/642 Now, we move onto a much more human discussion. We have all these powers in our reach, but should we use them? If yes, then how should we use them. Ethics of CRISPR/Cas9 application: 'With great power, comes great responsibility’ said Ben Parker to a young Peter as they drove to a library one evening. To those who are uninitiated, this is a direct quote from Spider-Man and is one of the most iconic lines in superhero literature. Uncle Ben said that to Peter Parker in order to teach him that any sort of power comes with a certain amount of responsibility and it is upto us to not abuse it or not to harm others, but to help them and make the world better a better place. This quote precisely explains the dilemma scientists face with this revolutionary technique. We can edit almost all gene segments, fi 25 Use of CRISPR/Cas beyond biological research: CRISPR/Cas9 has been applied extensively in biological research in order to understand the depths of the workings of biological systems. However, given that this technique is so exible, it has been applied in multifarious real world applications. To this day, many feats have been achieved using CRISPR/Cas9. Some of them include genetically engineered plants which boast greater resistance to stress (91,92) and increased carbon xation e ciency. (93) Variants of Cas9 have also been used to arti cially generate resistance against RNA based plant viruses. (103) Not just plants, CRISPR/Cas9, despite its aws, has been able to achieve incredible feats in the world of animal therapeutics. Duchenne Muscular Dystrophy (94), ⍺-1 antitrypsin de ciency (95), haemophilia (96) and even the battle against the dreaded HIV have all received contributions because of CRISPR/Cas9. (97) Recently, this system has also been used as a diagnostic tool to detect SARS-CoV-2. (98) Beyond these, it is expected that CRISPR/Cas9 will serve as an e ective weapon in the ght against some viruses which can lead to latent infections e.g. Epstein-Barr virus, John Cunningham virus etc. (103) Besides all these, cancer diagnostics have been revolutionised by the advent of CRISPR variant based techniques such as SHERLOCK. (99) Also, in the battle against cancer, positive results have been reported in mouse models where prevention of development of cancer was achieved by preventing gain of function mutations, (100) inhibiting loss of function mutations, (101) along with immunotherapy using genetically modi ed Chimeric antigen receptor T cells, all experiments which used CRISPR/Cas9. (102) In 2018, CRISPR was used by He Jiankui to edit human embryos and the rst CRISRP/ Cas based clinical trial to treat HIV was also set in motion. In 2019, in vivo clinical trials using CRISPR/Cas9 in order to treat blindness were authorised, signifying the giant leaps the technique has taken since its inception in 2012. (103) In coming years, the trend might shift towards more invasive germline editing procedures. But as we attempt to perform more and more invasive procedures, questions arise regarding natural laws we might be breaking. Is it really okay for us to mess with nature like this? What possible dangers might we be dealing with by editing embryos? And if we are editing human embryos and various other organisms, where do we draw the line? Before we deal with these questions, it is important to look at unauthorised uses of CRISPR, especially He Jiankui’s attempt at editing human embryos. Major misuse of the tool: On 27 November 2018, an entire sect of the scienti c community sat back in shock and awe. Their smartphones bombarded with one hashtag. The infamous hashtag that signi ed a line being crossed in the world of genetic editing which was supposedly the called the red line, as in, must never be approached, let alone crossed. #CRISPRbabies trended on twitter. For the rst time, someone had used CRISPR/Cas9 to directly edit the fi fi fi fi fl ff ffi fi fi fi fi 26 fi fl we can alter the course of evolution and we can manipulate not just DNA, but even RNA at our will. But should we do all that? Who should have access to a technology which in the wrong hands could lead to a catastrophe? Let us explore these questions together. What He Jiankui had just done was that he used a edgling and o target e ect prone technology to edit human embryos. Although he performed such an act to protect two children from the wrath of HIV, still, the scienti c community almost lashed out. People understood that to want to help someone in need is a noble quality, however, using an unruly tool whose e ects one cannot fully predict is not only irresponsible, but also verging on criminal. The Chinese judicial system agreed with the world's opinion on this one as Jiankui was sentenced to 3 years in prison and was slapped with a US $430,000 ne. His cohorts were also punished with prison sentences, albeit shorter ones. (105) No one questioned the fact that Jiankui intended only to help two kids so that they could live a normal life, but in doing so he endangered two human lives. If things had gone sour and some truly harmful o target snips had occurred, maybe two lives would have been lost. Fortunately, the two babies were born healthy, but this still does not exempt Jiankui of the act he committed. What redoubled his egregious act was the fact that Jiankui in his study started with 8 HIV positive couples. This experiment was largely regarded as a failure of ‘self-regulation’ in the world of biological science. (104,106) More problematic was the fact that this was not an isolated incident. Whispers had been swirling around gene editing in humans being performed in China since as early as 2015, but nothing became of them since the projects were kept highly classi ed. Some of these were revealed later on, one example being Huang’s attempt at editing embryos in 2015 in order to combat thalassemia. These projects were functioning even though the progenitor of the technique, Jennifer Doudna, strictly preached against such an application. She was right in doing so, since these projects reported o target e ects in the genomes of the embryos. So, any application of CRISPR/Cas9 to edit anything in the human genome could be potentially catastrophic. It could become as serious of a crime as manslaughter, since it is argued that any embryo beyond 14 days in age is essentially a human life. (54) Although the actions of He Jiankui were ill advised, his experiment did have a positive e ect. This experiment sparked a wave of debates which discussed on a global scale the ethics of application of CRISPR/Cas9 and at some level, the ethics of its distribution. He Jiankui himself contributed to these debates by mentioning 5 guidelines for CRISPR/Cas9 usage in human embryo editing 1. Empathy for patients - for some families, early gene surgery may be the only way to cure disease. 2. Only for serious disease, never for vanity. 3. No one can control a child’s life. A gene-edited child retains the same rights as a “normal” child. 4. Genes do not de ne us. DNA does not predispose us. 5. Everyone deserves freedom from genetic disease, regardless of wealth. The above guidelines are a direct quote from the book Editing humanity by Kevin Davies, which covers this entire asco in detail. (54) This gives us a lot of talking points to tackle. ff fi ff ff fl ff fi fi ff ff fi 27 ff fi genomes of twin babies, Lula and Nuna, in order to immunise them to HIV (CCR5 gene was targeted which is what HIV strains use to infect human cells). But, no one commended this action. In fact, the very next day, when the perpetrator He Jiankui walked on stage during the gene editing summit in Hong Kong, he was greeted with almost a disdain from his peers. Headlines poured from various news outlets, CRISPRbaby scientist fails to satisfy. (54,104) The nal conversation we shall have then will be discussing these guidelines and ethical conundrums. A large section of this debate shall be my personal opinion and the reader is free to agree or disagree with them. The ethical dilemma of genetic engineering in humans: Imagine a scenario. A couple walks to you and tells you that their child will be born with a debilitating disease. You possess a tool to solve their problems, but it is risky. It will not work as intended every time. What will you do? Will you argue that the unborn child has its own rights and you cannot perform a permanent procedure and change that life forever? What another argument would be is that, will you risk loss of life in order to improve the quality of life for an embryo? At this point you enter a whole other realm of philosophical thinking - is it better to be disabled and be alive or to not be alive at all? These arguments seem to have no end, even then, I shall humbly present my take on them. Should He Jiankui have done what he did? The simple answer is no. You simply do not take a risk this big, no matter what the situation may be because the risk does not lie with one generation. This is a permanent genetic change which will be inherited by subsequent generations, hence, one misstep may have doomed an entire line of descendants. So, it was a highly irresponsible decision that was spurred on by emotions rather than logic. Further, one might go so far as to say that Jiankui might have wanted to capitalise on the desperation of those couples, but with regards to this, I shall give him the bene t of the doubt. Sometimes you have to let something bad happen in order to avoid an eventuality which is much worse. A chess player would empathise with this analogy, and so would anyone who has lived long enough in this world which is not just roses and rainbows. So, even though what Jiankui did was bad, it did serve as a wake up call to all of us. Should humans be allowed to edit the genomes of other humans? If it is for genetic therapy in adults, an authorised clinical trial is where this such editing should happen. Of course, all those who are participating in the trial must be made aware of the risks they are taking. This is how medicine has worked for more than a century now. But where this should not happen for now is in germline cell gene editing. Despite one’s consent, germline editing is something which could adversely a ect the coming generations and thus the decision does not really lie with the person consenting to the CRISPR based therapy. Should this happen in embryos? It could be done for embryos but not fait accompli like what Jiankui did, who informed the world of his act after the fact. Again, such an act should be performed as part of a clinical trial, under strict observation and such embryos should not be implanted into a surrogate mother, so as to avoid risks to both the mother and the unborn child. Thus, such a clinical trial must be only for research purposes until a satisfactorily accurate method of gene editing is developed. Who should have access to CRISPR/Cas9 technology? This one is a touchy subject. As a scientist, one wants knowledge to be disseminated far and wide into the world, so that anyone in need of it can access it. However, nefarious individuals exist on this planet. ff fi fi 28 Many a times there have been instances where bio-weapons were used to terrorise innocent people. Powdered Bacillus anthracis comes to mind rst of all as I was warned multiple times as a child, not to open just any letter and in case there was some sort of powder in it, to contact the doctor at once. So, in a world where bio-weapons are already a reality, what could CRISPR/Cas9 editing do? It is not too far fetched to say that CRISPR based editing and knock-ins could create pathogenic strains with increased virulence and deleterious e ects, di cult though it may be. So, should anyone still have access to CRISPR/Cas9? I would still say yes. However, in an ideal world, CRISPR/Cas9 engineering would be taught to only trained scientists who would allow a higher level of transparency in their labs’ proceedings and it is via these scientists that both the knowledge regarding CRISPR/Cas9 and its application would be accessed and distributed. This does not mean that all the open access literature must be done away with. This only means that any lab working with CRISPR/Cas9 must open its door to investigations and become even more transparent as compared to a lab which does not. I understand that this could hinder a lab’s functioning and would make CRISPR a di cult subject to conduct research on, however, such measures must be enforced in order to avoid a nearly criminal act as what He Jiankui did. A lot of conversations and debates need to be had before we take any steps in the direction of human gene editing. I would like to point you to one such conversation with Jennifer Doudna, the creator of this technique. ffi fi fi ffi fl ff The conversation would be Jennifer Doudna’s interview at the World Science Festival in October 2019. (108) Interesting questions were addressed to her during her time on stage. How could CRISPR base editing improve one’s quality of life? Could it maybe increase a person’s life span? She answered by basically saying that CRISPR based therapy could not achieve an increase in a person’s life span because we do not understand ageing completely, but it could be used to prevent the shortening of lifespan by helping in therapy of illnesses such as cancer or by allowing humans to ght against infections. In another part of the interview, she addressed the question of designer babies. She again went on to say that achieving traits such as increased intelligence or increased athleticism could be under the combined control of thousands of genes, so realistically, achieving that still would not be possible. Same answer for super soldiers. So sadly, no Captain Americas for us in the near future. She also addressed notions such as bringing back species which had gone extinct in recent times. Or even organising gene drives, which she admitted were possible to some extent with the current state of CRISPR technology. She also took the time to discuss one of her articles in nature titled ‘Genome-editing revolution: My whirlwind year with CRISPR’ (107) an article which was prompted by the fear that CRISPR/Cas9 would be used to edit human embryos. As the interview continued, she discussed many a topic, but what she stressed most of all was the fact that it is important for us to have ethical debates regarding CRISPR and that these debates should go global so that we can educate people. I would urge the reader to go on YouTube and watch this video and thus introspect and have a conversation with themselves regarding CRISPR/Cas9. As for my introspection, I agree with Jennifer on all of the points. What I would like to add to all of this is that CRISPR is an important technology. It is not too far fetched to say that it could have the same implications as Alfred Nobel’s invention of dynamite, which I would like to remind the reader, was at one point intended to make roads on mountains by blowing away portions of it, but as we know now, the invention of dynamite instead led to the weaponisation of the world, an eventuality which has exacted devastating damages. In the same tune, CRISPR/Cas system has amazing therapeutic potential, however it could be dangerous because it can be learnt quickly, it is exible and it can also be applied quickly and thus, in nefarious hands, it could endanger the world. What can be taken away from all of this is that 29 managing CRISPR is not a scienti c responsibility alone, it is a global responsibility. In the future, there may come a time where CRISPR touches all of us intimately. It may become a part of our lives. So, we must develop international and national level regulatory infrastructures so that knowledge regarding CRISPR can be distributed in a safe manner in order to ensure that this gorgeous gift that biological sciences has provided us, does not get abused like many other scienti c gifts that came before it, because this time, we really cannot a ord any such mistakes. The bottomline therefore is this. CRISPR can be used in humans but only for therapeutic purposes under strict regulation. CRISPR for vanity is completely out of the question. The myriads of uses of this technology such as genetic engineering of crops, biological research, ghting against antimicrobial resistance and all other applications in the same vein must be encouraged. The world must start thinking about how to use CRISPR/Cas properly and how to prevent its misuse. Finally, decisions involving CRISPR/Cas must be made devoid of emotion, based on pure scienti c logic. Thus, a lot of responsibility rides on our shoulders. A few missteps here and there could doom an entire race. So, instead of shying away from it, we must face it with vigour. We must own up to the challenge. Humanity birthed this technology and thus all of humanity combined must raise this wonder child properly. Conclusion: Beyond all the heavy talk, to end this review on a positive note, I would like to add that a world where humans will have access to their own genome via CRISPR/Cas9 may not be too far away. A world where cancer is not a terminal malady but a mild disease which can be xed with a trip to the doctor’s o ce for a little hit of CRISPR/Cas9 may not be completely imaginary. A world where resistant bacteria do not pose major threats because we have a knight in shining armour, armed and ready to ght for us, may soon be upon us. Dreams of a scienti c utopia pop up whenever the future of CRISPR/Cas9 is mentioned. Which dreams shall we attain and which shall be lost in the annals of human endeavour, only time will tell. But what CRISPR/Cas9 most de nitely has done is reinvigorated the world of science by attracting a new generation of budding scienti c minds to the eld. In this review, we have explored the entire journey of CRISPR/Cas9 system. A simple sequencing mistake led to the discovery of CRISPR repeats and in the following years, the very repeats generated a technology which will de ne biological scienti c e orts in the coming century. To be a part of such an e ort, we understood the basics of the mechanism by which this system functions, followed by where it stands in the current world. Then we took a foray into the recent exciting variations that have been coming up in the past 5 years and discussed how these very developments shall lead us into the future. All of this scienti c text was underlined by a human discussion of the morality of CRISPR/Cas9 usage, a conversation which I would say is just as important as the scienti c aspect of this review, if not more. Hence, we arrive at the end of this beautiful ride. The sole purpose behind this review was to introduce the uninitiated to the world of CRISPR and to give them enough knowledge and ideas to set them on their way. Thus, such a large text was generated in pursuit of that goal. To conclude this text then what I would like to say is that millennia of scienti c thoughts, studies and experiments led us to this unbelievable discovery. It must therefore be treasured, applauded, disseminated and applied, albeit responsibly. fi ff fi fi fi fi fi ff fi fi ffi fi fi fi ff fi fi fi fi 30 I would like to thank Professor Saikrishnan Kayarat for providing me with this opportunity to review a technique which motivated to become a scientist. His advice and teachings have been invaluable as I wrote this review. Further, I want to thank IISER Pune for having such a course as Literature review which trains budding scientists in the art of writing scienti c literature. This review, although will not be published, shall always remain my rst piece of scienti c literature. References: History of CRISPR: 1. 2. -. 13. 14. Ishino, Y., Shinagawa, H., Makino, K., Amemura, M., & Nakata, A. (1987). Nucleotide Sequence of the iap Gene, Responsible for Alkaline Phosphatase Isozyme Conversion in Escherichia coli, and Identi cation of the Gene Product. In JOURNAL OF BACTERIOLOGY (Vol. 169, Issue 12). Van Soolingen,’, D., De Haas,’, P. E. 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