Genome editing is a type of geneticengineering where a DNA is inserted, deleted or replaced in the genome of aliving organism. The application of this technology has revolutionized variousresearch areas ranging from biomedicine to biotechnology or synthetic biology.A key point to initiate the editing is the need to generate a double strandbreak (DSB) in the DNA at a specific locus in the genome. To achieve thisprecise DSB researchers have developed engineered nucleases, also termed “molecularscissors”. Previous efforts have focused in the molecular understanding andredesign of different protein templates, such as homing endonucleases, zincfinger nucleases (ZFN) and TALEN. These tools have shown their utility indifferent genome editing applications including the correction of mutationsinvolving monogenic diseases. However, the engineering of new DNA specificitiesin these protein scaffolds is cumbersome. Therefore, the development of theversatile CRISPR-Cas systems (Clustered Regularly Interspaced Short PalindromicRepeats-CRISPR associated proteins) as molecular scissors, where a simpleexchange of the RNA guide sequence is enough to redesign the nucleasespecificity has paved the way for a revolution in the life sciences.

CRISPR repeats are associated with Casproteins constituting an adaptive immune system in bacteria and archaeaprotecting them from foreign mobile genetic elements . The discovery ofCRISPR-Cas revealed the capability of bacteria and archaea to acquire andintegrate genetic elements into its own genome, demonstrating the exchange ofinformation between the environment and prokaryotic genomes. The genetic recordof previous attacks by foreign nucleic acids is stored in the CRISPR arrays.These arrays are made of short and conserved repetitive sequences calledrepeats which are strategically placed between unique sequences called spacers.They are inserted by specialized Cas proteins into the CRISPR array duringinfections by invading nucleic acids. The adaptive immunity by prokaryotesagainst foreign MGEs is achieved through the formation of RNA-guidedendonucleases, which constitute the effector complexes and are able to detectsecondary infection by a foreign DNA that was previously incorporated into theCRISPR array.

The CRISPR-Cas systems are classified intotwo classes (Classes 1 and 2) that are subdivided into six types (types Ithrough VI). Class 1 (types I, III and IV) systems use multiple Cas proteins intheir CRISPR ribonucleoprotein effector nucleases and Class 2 systems (typesII, V and VI) use a single Cas protein . Class 1 CRISPR-Cas systems are mostcommonly found in bacteria and archaea, and comprise ∼90% of all identified CRISPR-Cas loci. The Class 2 CRISPR-Cassystems, comprising the remaining ∼10%, exists almost exclusively in bacteria , and assemble aribonucleoprotein complex, consisting of a CRISPR RNA (crRNA) and a Cas protein. The crRNA contains information to target a specific DNA sequence . Thesemultidomain effector proteins achieve interference by complementarity betweenthe crRNA and the target sequence after recognition of the PAM (ProtospacerAdjacent Motif) sequence, which is adjacent to the target DNA . Theseribonucleoprotein complexes have been redesigned for precise genome editing byproviding a crRNA with a redesigned guide sequence, which is complementary tothe sequence of the targeted DNA . The most widely characterised CRISPR-Cassystem is the type II subtype II-A that is found in Streptococcus pyogenes(Sp), which uses the protein SpCas9, Cas9 was the first Cas-protein engineeredfor use in gene editing . Class 2 type V is further classified into 4 subtypes(V-A, V–B, V–C, V–U). At present, V–C and V–U remain widely uncharacterised andno structural information on these systems is available . V-A encodes theprotein CRISPR Cas12a protein (also known as Cpf1) and recently several high resolutionstructures of Cas12a have provided an insight into its working mechanism .

This system, involving RNA-guided interference, has been harnessed into a versatile biotechnological tool for genome editing, whereby a simple exchange of the RNA guide sequence can be employed to re-engineer nuclease specificity, leading to a revolution in the life sciences. Cas9, belonging to Class 2 Type II CRISPR-Cas interference system, is the more extensively used tool for genome editing. However, the overwhelming efficiency in genome sequencing of different organisms has generated a large amount of data helping the identification of new systems whose use in genome editing is currently being explored. Among them there are new members of Class 2, such as Cas12a.

CRISPR-Cas immunity involves three major sequential steps: adaptation, expression/maturation and interference (Fig. 1), each step needs specific Cas proteins encoded by the cas genes near the CRISPR array, together with other accessory proteins. The CRISPR-Cas adaptation stage involves the identification and extraction of the protospacer from the invading DNA/RNA and its subsequent incorporation into the CRISPR array. Both these functions are performed by the versatile Cas1-Cas2 adaptation complex. The identification of the protospacer starts with the recognition of the PAM by the adaptation complex, subsequently the spacer (sequence adjacent to the PAM) is integrated into the CRISPR array and the conserved repeat sequence is duplicated. The PAM sequence is excluded from the CRISPR array and is one of the first recognition motifs used for identifying target nucleic acids for degradation. During the expression/maturation stage, the CRISPR array is transcribed into a long pre-CRISPR RNA (pre-crRNA) molecule. The pre-crRNA is processed into shorter crRNA molecules each containing a spacer and a part of the repeat sequence. Finally, interference can occur, after the crRNA forms a complex with the effector protein, forming a functional RNA guided endonuclease. This endonuclease is guided by the crRNA, which after PAM recognition hybridizes with the target DNA through its spacer sequence, and eventually, cuts the target DNA sequence. In this review, we describe the structural and functional features of Cas12a, a cousin of Cas9 belonging to the Class 2 Type V CRISPR-Cas system, which has been repurposed into an alternative and promising gene editing tool based on its substantial differences with Cas9.

Fig 1. Stages of CRISPR-Cas immunity- Adaptation, Expression/Maturation, Interference.

CRISPR Cas12a crRNA biogenesis

RNA sequencing of small RNA moleculesextracted from Francisella novicida U112 culture containing Cas12a-based CRISPRloci revealed that mature crRNAs for Cas12a protein are 42–44 nt in length, with thefirst 19/20 nt corresponding to the repeat sequence and the remaining 23-25 ntto the spacer sequence . In type II CRISPR systems, the maturation of crRNA isdone by host housekeeping protein RNase III together with the trans-activatingcrRNA (tracrRNA), which is base paired with the pre-crRNA, in presence of Cas9 .In contrast, it has been shown that Cas12a processes its own pre-crRNA intomature crRNAs, without the requirement of a tracrRNA, making it a uniqueeffector protein with both endoribonuclease and endonuclease activities .Afterthe pre-crRNA has been transcribed during the expression stage, Cas12a cuts it4 nt upstream of the hairpin structures formed by the CRISPR repeats, producingintermediate crRNA molecules which undergo further processing in vivo intomature crRNAs.

CRISPR Cas12a domain organisation andribonucleoprotein complex assembly

Type II (Cas9) and type V (Cas12a)CRISPR-Cas systems possess a characteristic Ruv-C like nuclease domain (Fig.2A), which has been shown to be related to IS605 family transposon encoded TnpBproteins . Crystallographic and cryo-EM data   reveal that Cas12a protein adopts a bilobed structureformed by the REC and Nuc lobes (Fig. 2B). The REC lobe is comprised of REC1and REC2 domains, and the Nuc lobe is comprised of the RuvC, thePAM-interacting (PI) and the WED domains, and additionally, the bridge helix(BH). The RuvC endonuclease domain of this effector protein is made up of threediscontinuous parts (RuvC I-III). The RNase site for processing its own crRNAis situated in the WED-III subdomain, and the DNase site is located in theinterface between the RuvC and the Nuc domains. These structural studies havealso shown that the only the 5’ repeat region of the crRNA is involved in theassembly of the binary complex. The 19/20 nt repeat region forms a pseudoknotstructure through intramolecular base pairing. The crRNA is stabilized throughinteractions with the WED, RuvC and REC2 domains of the endonuclease, as wellas two hydrated Mg2+ ions . This binary interference complex is thenresponsible for recognizing and degrading foreign DNA.

Fig2. Domain organisation and R-loop complex of Ca9 and Cas12a..

PAM recognition

PAM recognition is a critical initial stepin identifying a prospective DNA molecule for degradation since the PAM allowsthe CRISPR-Cas systems to distinguish their own genomic DNA from invadingnucleic acids . Cas12a employs a multistep quality control mechanism to ensurethe accurate and precise recognition of target spacer sequences. The WEDII-III, REC1 and PAM-interacting domains are responsible for PAM recognitionand for initiating the hybridization of the DNA target with the crRNA. Afterrecognition of the dsDNA by WED and REC1 domains, the conserved loop-lysinehelix-loop (LKL) region in the PI domain, containing three conserved lysines(K667, K671, K677 in FnCas12a), inserts the helix into the PAM duplex withassistance from two conserved prolines in the LKL region. Structural studiesshow the helix is inserted at an angle of 45° with respect to the dsDNAlongitudinal axis, promoting the unwinding of the helical dsDNA. The criticalpositioning of the three conserved lysines on the dsDNA initiates theuncoupling of the Watson–Crick interaction between the base pairs of the dsDNAafter the PAM. The target dsDNA unzipping allows the hybridization of the crRNAwith the strand containing the PAM, the ‘target strand (TS), while theuncoupled DNA strand, non-target strand (NTS), is conducted towards the DNasesite by the PAM-interacting domain . Cas12a has been shown to efficientlytarget spacer sequences following 5’T-rich PAM sequence. The PAM for LbCas12aand AsCas12a has a sequence of 5′-TTTN-3' and for FnCas12a a sequence of5′-TTN-3′ and is situated upstream of the 5'end of the non-target strand . Ithas also been shown that in addition to the canonical 5′-TTTN-3′ PAM, Cas12aalso exhibits relaxed PAM recognition for suboptimal C-containing PAM sequencesby forming altered interactions with the targeted DNA duplex .

DNA unzipping, propagation and cleavage

Once the crRNA-DNA hybrid R-loop startsforming, the enzyme then looks for a seed sequence of 3-5 nt on the PAMproximal end, the next check point in the correct identification of the target(Fig. 3). It has been reported that mismatches in the seed sequence results inthe loss of cleavage activity . Presence of the seed sequences promotes furtherhybridization of the crRNA-target DNA. Structural studies have shown that TSand NTS follow different pathways to the nuclease site , with several residuesin the PI domain undergoing conformational changes and adopting a ‘rail’ shapeto accommodate the nt-strand and eventually guiding it to the catalytic site.This structure also shows the presence of a barrier, the septum, to prevent there-annealing of the dsDNA.

Fig3.   Model mapping the catalytic pathway of CRISPR-Cas12a.

A recent cryo-EM analysis on theintermediate catalysis products of Cas12a revealed that the enzyme utilizes afurther three-checkpoint control to sense the hybridization between the crRNAand the DNA. Three regions within the enzyme, the ‘REC linker’, the ‘lid’ andthe ‘REC finger’ sequentially scan the hybrid through conformational changesand only when all three checkpoints are able to recognize the hybrid, theenzyme is in a conformation competent for catalysis. The endonuclease producesa staggered cut on a PAM distal site on the DNA with a 5 nt overhang on thetarget strand , and the PAM distal end of the cleaved product is then releasedfrom the complex .

Cleavage in the t-strand of the DNA byCas12a produces a 5′-phosphorylated product . In order for both DNA strands tobe cut, they must enter the catalytic site with a 5′-3′ polarity. Structuralstudies reveal that the NTS is positioned to enter the RuvC-Nuc pocket with the5′-3′ polarity, while the TS has the reverse polarity. An smFRET analysissuggests that CRISPR Cas12a has to undergo conformational changes in the distal partof the REC and NUC lobes in order to allow the TS enter the nuclease site withthe correct polarity . This could explain why the NTS appears to be hydrolyzedfaster than the TS. Therefore, the cleavage of the NTS is a consequence of theproper positioning of this strand in the RuvC-Nuc catalytic pocket rather thana requirement to initiate the cleavage reaction. After both strands have beencleaved, the PAM distal end of the cleavage product dissociates from thecomplex, but the PAM proximal site remains associated to Cas12a forming acleaved R-loop .

Indiscriminate ssDNA cleavage

Besides high-specific dsDNA cleavage,Cas12a has also been shown to exhibit indiscriminate ssDNA degradation activityupon activation with a ssDNA complementary to the crRNA guide. This activity isdisplayed by all Cas12a orthologs and degrades any available ssDNA moleculeinto single/double nucleotides . Comparisons of the structures of Cas12abefore, during and after cleavage reveal the structural changes that result insuch an indiscriminate activity. The lid region, which is involved in thecheckpoints for accurate target recognition is responsible for this action.Before the crRNA-DNA hybrid is formed, the lid occludes the cleft where thecatalytic residues reside. Upon formation of the hybrid, the lid changesconformation to form an α helix, thus interacting with the crRNA of the hybridassembly, thus dissociating the polar interactions and making available thecatalytic pocket. In the R-loop structure after cleavage , this region appearsdisordered indicating that the catalytic site is accessible after the distalpart of the dsDNA substrate dissociates from the complex. Therefore, thecatalytic cleft is open and able to sever ssDNA indiscriminately. This molecularmechanism would explain how ssDNA molecules are degraded by Cas12a after beingactivated by the presence of the RNA-DNA hybrid. In addition, recent studieshave reported non-specific nicking of target sequences bearing mismatches indistal regions of the target DNA , suggesting that this could be a problem forpotential applications.

Cas12a endonuclease recycling

Unspecific ssDNA degradation presents apotential harmful situation for the host cell since it could hinder basiccellular processes such as replication, transcription and DNA repair. Thisposes an important question: how could we eliminate this harmful indiscriminateactivity? As the cell cannot allow an indiscriminate ssDNA degradationunleashed. The answer to these problems can be found in the bacterial genomesencoding Cas12a. Use of a conserved sequence of the crRNA for a database searchdisclosed that different bacteria encode a single copy of the Cas12a gene,whereas they encode multiple copies (up to 68) of the crRNA . If thetranscription rates are presumed to be similar, at any given time, theconcentration of various crRNAs in the cell would be multiple times higher thanthat of the enzyme. It has been experimentally shown that with sufficientconcentration of a new crRNA molecule, it is able to displace the cleavedR-loop from the enzyme, with the help of accessory host proteins, forming a newinterference complex (Fig. 3). This shows that Cas12a can revert the activeconformation to shut down unspecific activity by displacing the cleaved R-loopwith a new crRNA. In doing so, it reverts back to a conformation where themolecular ‘lid’ forms polar interactions again to make the catalytic pocketinaccessible . By doing so, not only does the endonuclease shut down theindiscriminate ssDNase activity, but also recycles its catalytic activitytowards other target DNAs.

What is different between Cas12a and Cas9 ?

Cas12a and Cas9 have striking functionalsimilarities despite having evolved through independent pathways (Fig. 2, Fig.4), with similar sizes (1368 amino acids for SpCas9; 1307 for FnCas12a). Theyare both multidomain effector proteins and adopt a bilobed architecture when incomplex with their respective RNAs. Cas9 requires two RNA molecules: tracrRNAand a crRNA, whereas Cas12a requires only a single RNA molecule, the crRNA.Cas9 possesses two nuclease sites HNH and RuvC domains, while Cas12a possessesonly one nuclease site in the RuvC domain. Additionally, Cas12a also possessesan RNA processing site. There are distinct differences in the mechanismsemployed by the two proteins when it comes to RNA processing, PAM recognition,target DNA binding and eventually catalysis.

Fig4. Cas12a vs Cas9.

After the CRISPR array has been transcribedinto a long pre-crRNA molecule, it is processed into mature crRNAs before itcan form an RNP with the endonuclease. In the case of Cas9, the tracrRNA(encoded close to the CRISPR locus) first needs to hybridize with thepre-crRNA, and then this hybrid RNA–RNA duplex structure is recognized by Cas9,following which the host RNase III cleaves the duplex, leaving a ∼75 nt long tracrRNA and a 39-42 nt long crRNA, which then forms theRNP complex responsible for recognizing and degrading the target DNA . Incontrast, Cas12a does not require a tracrRNA or RNase III, since the proteinprocesses its own crRNA in its ribonuclease catalytic site . In type-V CRISPRlocus of F. novicida, the spacers are 27–32bp sequences interspersed by 36bprepeat sequences in between the spacers. In the entire CRISPR array transcript,which is the pre-crRNA, the repeat derived sequences form pseudoknots, whichare recognized by Cas12a. Following the recognition, the pre-crRNA is cleavedforming ∼43 nt maturecrRNA . Logistically, Cas12a presents a more minimalistic system than Cas9.

For Cas9 targeted DNA sequences, the PAM issituated downstream of the spacer sequence on the non-template strand, and isrecognized by the PI domain, which is primed for identifying a 5′-NGG-3′ PAM .In contrast, Cas12a recognizes A-T rich sequences, with the PAM, typically5′-TTTV-3′, located upstream of the spacer. Upon PAM recognition, the targetDNA is unzipped and hybridization of the RNA-DNA takes place. For both enzymesexists a crucial seed sequence next to the PAM to determine the specificity oftarget DNA binding. The seed sequence for Cas9 is about ∼10 nt whereas for Cas12a it is about ∼5–6 nt . When the hybridization of the DNA with the RNA is complete,Cas9 cleaves the template strand and the non-template strands in the catalyticsites located in the HNH and the RuvC domains respectively, producing a bluntDSB, with the cleavage site being 3 base pairs upstream from the PAM sequence .However, in the case of Cas12a due to the presence of a single nuclease site,the strands of the DNA are cut in the same nuclease site. Since it has beenshown that the two strands follow different pathways to reach the catalyticsite, explaining the staggered DSB produced by Cas12a .

Cleavage fidelity is an important issue formany of the nucleases used in genome editing applications . An optimal toolmust introduce modifications just on the target site, leaving the rest of thegenome unmodified in order to avoid undesired changes in other sites of thegenome with unpredictable consequences. Therefore, specificity and theresulting cleavage products are key in genome modification applications. Arecent comparison of different Cas12a and Cas9 from different species usingnuclease digestion and deep sequencing (NucleaSeq) in vitro, revealed that bothenzymes share similar types of specificities and tolerate similar mismatches ,in contrast to in vivo reports that show the lower off-target effects of Cas12a. This apparent contradiction may be related to different recognition andcleavage kinetics, but also to a possible different behaviour of these cutterson a chromatin context, thus posing the question whether in vitro or in vivoapproaches should be pursued for nuclease redesign efforts.

CRISPR-Cas12a mediated genome editing

Application of CRISPR-Cas systems asmolecular tools for genome editing exploits their ability to produce a doublestrand break (DSB) at a specific genomic locus, and depends entirely on thehost cell DNA repair machinery to fix the lesion produced by these systems. Therepair mechanisms can be either of the following processes: homology-directedrepair (HDR) or non-homologous end joining (NHEJ). HDR utilizes a template DNAthat is homologous to the break site (an unbroken sister chromatid or a homologouschromosome) to repair the DSB, whereas NHEJ is based on direct joining ofbroken ends of the DSB, making NHEJ the more error prone mechanism of the two.HDR can thus be used to supply exogenous template DNA to implement a userdefined change in the host genome. NHEJ can be applied for gene disruptionwhereas HDR allows for the scope of introducing new genetic information ordirect correction of the sequence at a specific locus.

At the center of CRISPR mediated genomeengineering today is Cas9, with applications including, but not limited to,gene knockout and precise genome editing. Despite the rapid advances in genomeediting by Cas9, it still presents challenges owing to the possibility ofoff-target effects and difficulty of delivering the ribonucleoprotein particle .Cas12a, owing to its substantial differences with Cas9, presents an alternatemolecular genome editing tool. The use of Magigen Cas12a in genome editing for variouscell types has been probed in several studies up to date. Comparative studies ofgene repression by catalytically dead Cas9 from S. pyogenes (SpdCas9) andcatalytically dead Cas12a from Eubacterium eligens (EedCas12a) revealed thatthe latter displays a higher gene repression in the template strand of thetarget DNA than SpdCas9 . It was also shown that the pre-crRNA processingactivity of Cas12a makes it an attractive candidate for multiplex generegulation, which is cumbersome when attempted with Cas9 . This auto-processingof its own crRNA has been used to modify multiple genetic elementssimultaneously generating constitutive, conditional, inducible, orthogonal andmultiplexed genome engineering of endogenous targets using multiple CRISPR RNAsdelivered on a single plasmid .

The viability of this approach has been furtherestablished by other studies, in which multiplex gene regulation by Cas12a wassuccessfully observed in bacteria, plants, as well as in mammalian cells .Cas12a can also serve as a solution in cell types where use of Cas9 is toxic,such as in some industrial strains of Streptomyces .

Targeted mutagenesis in plants can also beachieved through co-expression of Cas12a and its cognate crRNA in vivo, as wasshown in rice. Additionally, it was also shown that the mutagenesis was moreefficient through the use of pre-crRNAs with full-length direct repeatsequences than with mature crRNAs . Efficient mutagenesis through delivery ofthe pre-assembled ribonucleoprotein (RNP) particle was also observed in soybeanand wild tobacco. The RNP was assembled from recombinantly expressed Cas12a andin vitro transcribed or chemically synthesized crRNAs .

Successful gene editing of mammalian cellsusing Cas12a include correction of mutations causing Duchenne musculardystrophy (DMD) in patient derived induced pluripotent stem cells (iPSCs) andin mdx mice, a popular model for studying DMD. Dystrophin expression wasreinstated in iPSCs after Cas12a-mediated gene editing, while in the mdx mice,corrections in the pathophysiological hallmarks of muscular dystrophy wereobserved . Delivery of the adenovirus vector with an AsCas12a expressioncassette yielded successful mutations in primary human hepatocytes fromhumanized mice with chimeric liver . Cas12a-mediated genome editing was alsoused to engineer rat models that mimic human atherosclerosis and this systemmay have potential applications in understanding early stage atherosclerosis .

All of the above studies how Cas12a can beengineered for various applications. Despite the numerous recent advances inthe application of Cas12a, there remain vast avenues of unexplored potential ofCas12a in terms of therapeutics and diagnostics.

What can we use Cas12a for ?

Cas12a applications in bioengineering

Currently, a vast effort is ongoing toredesign all these tools for biomedical and biotechnological applications.However, recent studies have envisioned the possibility of using CRISPR-Casnucleases in bioengineering of smart materials, for example hydrogels   These water-filled polymers are encapsulatedby DNA. In a recent study, Cas12a has been used to specifically degrade the DNAscaffold of DNA hydrogels, thus opening the possibility that this smart cuttercan be turned into a programmable device to deliver the cargo of DNA encagedhydrogels in a determined location at a certain time. The cleavage propertiesof Cas12a make it an ideal candidate to promote controlled delivery of thecargo. Although, application of these approaches and their combinations can benow envisioned by many researchers, the range of possibilities in differentareas is so large that it is beyond our imagination.

Conclusions

In this review we have sought to offer acondensed overview of the functionality of CRISPR-Cas12a, discussing thestructural and functional features of the different stages of the reactionpathway leading up to the catalysis of the target DNA, and eventuallydiscussing the applications of Cas12a in brief. Cas12a employs amulti-checkpoint mechanism to ensure precise targeting of DNA, which is adesirable property in a genome editing tool in order to have low off targeteffects. Although it has been shown that the indiscriminate ssDNA degradationof Cas12a could be shut down through the recruitment of a new crRNA molecule,it could still potentially harm the host cell targeted for genome modification.Modulation of this activity is necessary to achieve higher regulation andcontrol of Cas12a catalysis, and in turn to achieve a more robust genomeediting tool. In this direction structural information has been used to redesignCas12a obtaining variants without ssDNA unspecific activity, thus severing onlydsDNA specifically .

Currently, Cas9 and Cas12a, are the solemembers of the CRISPR family that have been utilized for genome editing. Owingto their significant similarities and differences, just these two endonucleasesbetween themselves have made the applications of CRISPR highly versatile.Cas12a, in some cases, offers certain advantages over Cas9, for example in itscapability to be used for multiplex genome editing and production of staggeredDSB, which promotes HDR instead of NHEJ. Significant research also is ongoingto engineer artificial variants of Cas9 and Cas12a to recognize different PAMthan the wild type proteins, which will facilitate the targeting of a widerlibrary of genomes.

The rapid advent of the CRISPR-Castechnology for genome manipulation has been revolutionary for life sciences.Despite the vast application areas of this technology, the current state of theart of the CRISPR molecular tools (Cas9 or Cas12a) suffers from one importantdrawback: dependence on host cell DNA repair machinery. Both Cas9 and Cas12abased technology produce a double strand break (DSB) in the target DNA, andthis break is then repaired by endogenous DNA repair machinery with or withoutthe presence of a template. Although these tools have been successfullyutilized to obtain precise insertion of DNA into the targeted genomic loci,their efficiency differs from cell type to cell type . DNA repair through HDRis also related to active cell division, which makes these tools ineffective incell types that are not actively dividing, such as neurons. Recent studiescharacterizing CRISPR-associated transposase (CAST), which comprises Tn7-liketransposase subunits and a CRISPR effector from type V–K, could pave the way tonew avenues of gene editing using CRISPR systems since these systems areself-sufficient in precise DNA insertion and do not depend on endogenous cellDNA repair machinery . However, a large ongoing research is aiming to tailorboth Cas9 and Cas12a further to ensure precise DNA insertion into the targetedgenome. Even apart from this apparent drawback, both these tools have a vastrange of applicability and ongoing efforts are striving to produce improved andmore robust engineered genome editing tools.

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