Comparative Analysis of CRISPR Systems

Research will be limited to:
- Timeframe: 2023-2026 (Inferred from the date March 12, 2026)
- Method: CRISPR-Cas9 and Cas12a genome editing
- Topic: PAM guide RNA cut mechanism
- Topic: Off-target rates
- Topic: Applications

Strategy: I will conduct a comparative analysis of CRISPR-Cas9 and Cas12a focusing on recent advancements (2023-2026). The research will specifically target structural differences in PAM recognition (NGG vs. TTTN), guide RNA architecture (tracrRNA/crRNA vs. crRNA only), and the resulting cleavage patterns (blunt vs. staggered cuts). Furthermore, I will investigate recent high-throughput off-target profiling data and the latest clinical/industrial applications unique to each system.
Queries:
- CRISPR-Cas9 vs Cas12a PAM recognition mechanism 2023..2026
- Cas9 vs Cas12a off-target rates comparative study 2024..2026
- Cas12a staggered cut vs Cas9 blunt cut applications 2023..2026
- CRISPR-Cas12a guide RNA engineering vs Cas9 2025 2026
- Cas9 and Cas12a clinical trial applications comparison 2023..2026

Executing 5 concurrent searches:
- CRISPR-Cas9 vs Cas12a PAM recognition mechanism 2023..2026
- Cas9 vs Cas12a off-target rates comparative study 2024..2026
- Cas12a staggered cut vs Cas9 blunt cut applications 2023..2026
- CRISPR-Cas12a guide RNA engineering vs Cas9 2025 2026
- Cas9 and Cas12a clinical trial applications comparison 2023..2026

Found 6 papers. Key excerpts:

A Review on the Mechanism and Applications of CRISPR/Cas9/Cas12/Cas13/Cas14 Proteins Utilized for Genome Engineering. — V Edwin Hillary et al., 2023

Genome editing or gene editing is a popular technology used in medicines, therapeutic drugs, infectious studies, and agricultural biotechnology. The genome-editing tools have been employed to study the precise function of a gene by cutting and altering at a programmed locus through insertion, deletion, or replacement of targeted bases. In the beginning, conventional gene-editing techniques like homologous recombination (HR) were utilized for gene inactivation, but the effectiveness of HR was extremely low with the labor-intensive process [ 1 ]. Later, targeted gene knock-down utilizing RNA interference (RNAi) technique has provided researchers with rapid and low-cost technology to silence the gene of interest to study its functions. However, it also could not completely knock-down the targeted sequence, which faced unpredictable off-target effects and delivered only temporary or partial inhibition of gene function [ 2 ].
The genome-editing technique needs programmable sequence-specific endonucleases to produce the site-specific single-stranded breaks (SSBs) or double-stranded breaks (DSBs) at the targeted site that allow the endogenous repair mechanisms to fill the breaks [ 3 ]. These breaks are fixed by either of the two major repair mechanisms, (1) homology-directed repair (HDR) and (2) non-homologous end-joining repair (NHEJ) [ 4 ]. To facilitate specific DNA breaks, various genome-editing tools are developed previously, such as meganucleases or homing endonucleases [ 5 ], zinc-finger nucleases (ZFNs) [ 6 ], and transcription activator-like effector nucleases (TALENs) [ 7 ]. But these tools demand laborious efforts for cloning and protein construction to make DSBs, which hinders these tools from routine applications of genome editing. In 2012, the researchers developed clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) system-based genome editing tools [ 8 ]. CRISPR/Cas system mediates diverse adaptive immune systems against phages or plasmids. Due to unique features of simplicity in design, cost-effectiveness, and labor intensity, the research community has immediately adopted the CRISPR/Cas system as a user-friendly and robust RNA-guided DNA targeting tool for genome editing in various species [ 8 – 11 ]. Based on the mechanism, the CRISPR system

has been divided into two main classes (1 and 2) and six types (I-VI) [ 12 ]. In these, types I to III were extensively studied, whereas types IV and VI were recently discovered. Types I, II, and V cut DNA, type VI cleaves RNA, type III cleaves both DNA and RNA and the cleavage activity of type IV has not yet been identified [ 13 ].
Cas9 protein has been utilized for diverse applications such as fluorescent imaging, base-editing, and transcriptional activation, apart from targeted cleavage of dsDNA. Like Cas9, the Cas10 protein is also involved in various applications such as fluorescent imaging, base-editing, and RNA tracking. As an alternative to Cas9 and Cas10, the Cas12 protein enhanced genome-editing efficiency by targeting only T-rich motifs without utilizing tracrRNA. So Cas12 system has expanded editing applications such as base-editing and detecting transcriptional variations. Cas13 protein is also used for diverse applications such as imaging, base-editing, and detection of transcriptional variations. Recently, the Cas14 protein has advanced genome-editing efficiency without needing an adjacent protospacer motif (PAM) and performs transcriptional regression and base-editing. This review describes the Cas variants of two classes of CRISPR systems used for genome editing.
A group of researchers detected CRISPRs by analyzing the alkaline phosphatase gene, which is liable for the isozyme conversion of alkaline phosphatase ( iap ) in the E. coli K-12 strain [ 14 ]. They identified a genomic region that contains a series of 32 nucleotides of distinctive sequences flanked by invariable palindromic repeats on the 3′ end of the iap gene [ 14 ]. Later, distinctive parallel sequences were found in the other E. coli strains and Enterobacteria ( Shigella dysenteriae and Salmonella enterica ) [ 15 ]. Similarly, during the study of Mycobacterium tuberculosis strains, researchers identified 36 bp repeats interspaced with unique spacers of 35–41 bp [ 16 ].
In subsequent works, the CRISPR array was found in archaea ( Haloferax mediterranei and Streptococcus thermopile ), and the same was identified in 90% bacterial and 40% archaeal genomes [ 17 ]. However, this odd genomic sequence first

turned out to be the outline of the CRISPR array. Still, due to the absence of necessary genome sequence data, the biological function of CRISPR remained elusive. Jansen et al. in 2002 described the CRISPR-associated genes [ 18 ]. Following this, several CRISPR/Cas genes were identified. In 2005, spacer sequence was identified in many genomes, and unique spacer regions were found within the CRISPR array [ 19 ]. These results showed that CRISPR is an adaptive immune system to defend prokaryotic cells against phage infection through the RNA-guided process.
The CRISPR system has been classified into two major classes. In the Class 1 system, the RNA-guided target cleavage needs several effector proteins, but the Class 2 system requires only one RNA-guided endonuclease to cleave the DNA sequences [ 12 , 20 ]. The class 1 system of CRISPR is divided into three types I, III, and IV, and the Class 2 system is divided into types II, V, and VI [ 21 , 22 ]. In the type I system, the CRISPR/Cas locus contains the Cas3 signature gene that encodes a large protein with a helicase to unwind DNA-DNA and RNA–DNA duplexes [ 23 ]. The type II locus encodes multidomain protein to target and cleave the dsDNA [ 8 ]. The type III CRISPR/Cas possesses the Cas10 signature gene, encodes a multidomain protein with palm domain to target, and cleaves ssDNA [ 24 ]. Type IV system contains CRISPR-associated splicing factor 1 (Csf1), which encodes a ribonucleic protein, but the detailed function of this system is yet to be identified [ 21 ]. Type V locus possesses Cas12 signature gene (known as CRISPR from Prevotella and Francisella 1 (Cpf1), C2c1 or C2c3 protein) encodes RuvC (an E. coli protein involved in DNA repair) domain, which cleaves both dsDNA or ssDNA [ 25 ]. Type VI contains Cas13 (C2c2) that encodes higher eukaryotes and prokaryotes' nucleotide-binding domain (HEPN), which cleaves ssRNA [ 26 ] (Table 1 ). Table 1 Classification of CRISPR/Cas

Development of plant cytosine base editors with the Cas12a system — Huanhuan Wang et al., 2023

CRISPR/Cas genome editing technology is revolutionizing plant research and crop breeding [1] . Typical Cas9 and Cas12a CRISPR tools produce DNA double-strand breaks (DSBs) to edit targeted genes. In plants, DSBs are repaired mainly via the error-prone nonhomologous end joining (NHEJ) pathway and often produce knockout mutations by introducing nucleotide deletions or insertions (InDels). Although precise mutations can be introduced from a donor template via the homology-directed repair (HDR) pathway, the process suffers from low efficiency, owing mainly to weak HDR activity and donor delivery barriers in plant cells [2] . Base editing is an alternative system for precisely engineering nucleotides without requiring DSB and donor templates. Base editors (BEs) share a similar structure, containing a catalytically impaired Cas endonuclease and a single-stranded DNA deaminase [3] . Most plant BEs have been developed from RuvC domain-inactivated Cas9 mutants [4] , [5] . These nickases activate a DNA mismatch-repair mechanism to excise the target strand complementary to the edited strand, thus increasing the installation frequency of desired base substitutions. Current BEs, including cytosine base editors (CBEs) and adenine base editors (ABEs), have been extensively optimized to achieve highly efficient conversion between C•G and T•A base pairs in the plant genome [4] , [5] . Recently, glycosylase base editors (GBEs) have been reported [6] , [7] , [8] to confer C•G-to-G•C transversion in crops and tree cells, but further improvements in accurate editing efficiency are still needed.
Cas12a is a class of well-characterized CRISPR RNA (crRNA)-guided nucleases that have some unique features. Unlike conventional Cas9, which normally induces 1–3-bp InDels proximal to the G-preferred protospacer-adjacent motif (PAM), Cas12a tools generate larger deletions distal to Cas9-inaccessible T-rich regions [9] . Because of its pre-crRNA processing capability, Cas12a has been exploited for simplified multiplexed genome modifications and RNA template-mediated HDR gene replacement in plants [10] . Although there is a lack of nickase mutants to

date, there have been some efforts to render Cas12a a specific base editing platform. Several CBEs were established from a catalytically inactive version of Cas12a [11] , [12] , [13] , [14] . A set of Cas12a-CBEs, BEACONs, show editing efficiency comparable to that of SpCas9-BEs in mammalian cells and induce base editing in mouse embryos and offspring [14] . In contrast, Cas12a CBE-mediated C-to-T conversions have not yet been reported in plants, although adenine base editing has been induced in rice and cotton at a few genomic targets with low frequencies [15] , [16] .
In this study, CBEs with various architectures were engineered from Cas12a. Efficient programmable base conversions were obtained with high product purity in transgenic rice. Our results suggest that Cas12a could provide an alternative platform for developing precise genome editing tools in plants.
To generate Cas12a-BE3, the D832A mutation was introduced into LbCas12a from pHUN611 [17] . The rAPO1 and UGI components were amplified from the eBE3 vector for assembly with dCas12a through Gibson cloning [18] . Anc689, evoFERNY, and A3Bctd were codon-optimized for rice expression (Supplementary sequences) and synthesized (Genscript, Suzhou, China). A3A from SpG-A3A was applied to generate the N57G mutation of eA3A [19] . Human RAD51 single-stranded DNA binding domain (DBD) was amplified from Nm2-hyBE3 [20] . In the hyperactive base editor (hyBEs) architecture, Cas12a-hyCBEs were built by sequentially assembling several deaminases, the DBD, and the dCas12a-UGI fragment. Gibson assembly was applied to fuse TadA8 from SpG-ABE8e and dCas12a [19] . Cas12a-ABE8e was obtained by addition of triplet copies of SV40 NLS at the 3′ terminus of dCas12a by direct PCR. Amplifications were performed with Q5 High-Fidelity DNA polymerase (NEB, Ipswich, USA). A site-

directed mutagenesis kit was applied to precisely induce point mutations (Transgene, Beijing, China). An NEBuilder HiFi DNA Assembly kit was used for fragment assembly. The cloned, mutated and assembled sequences of the components or full-length BEs were confirmed by Sanger sequencing before further vector construction (Sangon Biotech, Shanghai, China). Cas12a-BEs were then separately inserted into the pHUC backbone via Pst I/ Sac I double digestion. To build an expression cassette of the crRNA, a CmYLCV promoter was cloned from pRN120 (Addgene #160696) [21] . The tRNA and HDV ribozymes were separately designed in the long primers of a spectinomycin resistance (SpR) gene. Finally, the promoter, tRNA-SpR-HDV element and a poly T terminator were assembled as the crRNA expression component and inserted into the pHUC-Cas12a-BE vectors by Hin dIII digestion. To construct Cas12a-BEs for specific genome targets, the forward and reverse oligos of the 23-nt guide RNA sequence were annealed. Together with the 4n96-type crRNA scaffold, the crRNA was inserted into the binary vector to replace SpR via the Bsa I-mediated GoldenGate cloning method. Following previously described procedures [22] , crRNA-containing clones were selected positively with kanamycin and negatively with spectinomycin and then confirmed by Sanger sequencing.
The vectors were introduced into Agrobacterium strain EHA105 via the freeze–thaw method [23] . The clones were identified by sequencing the regions of crRNA and base editor before plant transformation.
Rice transformation was performed following a previously established protocol with modifications [24] . Mature rice seeds were dehulled and sterilized. The embryos were isolated to induce calli for 3 to 4 weeks. After infection with Agrobacterium , 350–400 solid and yellowish calli per transformation were selected under 50 mg L −1 hygromycin pressure for 4 weeks. Approximately 200 resistance events were selected for plant regeneration. Only one plant from an independent event was chosen for rooting and genotyping. All plant materials were grown at 28 °C with a 16 h light and 8 h dark lighting cycle.
To quantify the editing

Current and Prospective Applications of CRISPR-Cas12a in Pluricellular Organisms. — Shaheen Khan et al., 2023

Clustered regularly interspaced palindromic repeats (CRISPR) are loci present in nearly all archea and half of bacteria species [ 1 ]. They are composed of repeats and spacers, the latter being inherited from phages or foreign plasmids. A spacer with part of the surrounding repeat sequences can be transcribed under the control of infection-induced regulators such as LeuO [ 2 ] and processed into a CRISPR RNA (crRNA) able to assemble with CRISPR-associated (Cas) proteins. Cas proteins have an endonuclease activity (generally DNase) and are subdivided in six types. Types I, III, and IV correspond to multi-subunit effector complexes, while types II, V, and VI gather single-subunit effectors [ 1 , 3 ].
In prokaryotes, CRISPR systems play the role of adaptive immune systems against bacteriophage infections. A segment of the crRNA termed guide RNA (gRNA), transcribed from a CRISPR spacer, hybridizes to its complementary sequence on the target nucleic acid, which is then cleaved by the Cas nuclease. The cleavage is performed only if the target sequence includes a protospacer-adjacent motif (PAM), which is necessary for the binding of the Cas protein on DNA and the hybridization of the crRNA.
SpCas9 was the first Cas nuclease to be used artificially in 2012 [ 4 ]. More recently, other Cas proteins have been adapted for artificial use, including non-Cas9 proteins [ 5 ]. Different CRISPR systems have been optimized for a wide range of applications spanning from base editing to gene editing, silencing, or activation. They have been used in vitro, in bacteria, or in cell cultures, but also in pluricellular organisms [ 6 ]. The use of CRISPR systems in plants and animals may facilitate crop or farm animal improvement and developments in medicine. Nevertheless, it raises unique challenges such as vectorization, species particularities in genome accessibility or repair, or cell-type diversity [ 6 , 7 ].
Cas12a or Cpf1 (CRISPR from Prevotella and Francisella ), is a type V-A Cas protein present in a few dozen bacteria species [ 8 ]. In 2015, a screen of 16 Cas12a proteins revealed that Cas12a systems are suited for gene editing and that

the Cas proteins of Acidaminococcus sp . (AsCas12a, or AsCpf1) and Lachnospiraceae bacterium (LbCas12a or LbCpf1) are the most efficient in human cells [ 8 ]. Since then, Cas12a has been increasingly used artificially [ 9 ]. The most frequently used orthologs are LbCas12a, AsCas12a, and FnCas12a. In plant cells, numerous other Cas12a orthologs proved highly efficient for gene editing, such as MAD7 [ 10 ], TsCas12a, ErCas12a, or Mb2Cas12a [ 11 ].
Cas12a displays unique features, such as higher specificity and ability to process crRNA arrays, turning it into an attractive alternative to Cas9. In this article, we comprehensively review the applications of Cas12a in plant and animal living organisms and discuss for which purposes it is most suited and which challenges it will face in future developments.
In this section, we discuss how Cas12a DNase activity can be harnessed for gene editing and more, using examples from pluricellular organisms.
As other Cas nucleases, Cas12a produces double-strand breaks (DSB) in DNA sequences complementary with its gRNA. These targeted DSBs can be harnessed for several applications of gene editing. Knock-outs: In higher eukaryotes, non-dividing haploid cells and eukaryotic cells in the G1 phase of the cell cycle, the main DNA repair pathway is non-homologous end joining (NHEJ) [ 12 ], a relatively error-prone pathway. Thus, a sequence targeted by CRISPR-Cas12a can be cut repeatedly by the nuclease until the repair mechanisms introduce indels (Fig. 1 ). When these mutations occur in exons, they can induce frame-shifts or less frequently insert stop codons or disrupt essential amino acids or splicing sites [ 7 ]. In each case, the protein produced by the targeted gene is likely to be inactivated or may not be synthesized due to non-sense mediated decay. Alternatively, introducing DSBs at two loci simultaneously can lead to the deletion of the sequences located between them. This process can be harnessed to knock-out kilobase to megabase-long sequences [ 10 , 13 ]. Knock-ins: In

certain cells, a DSB can be resolved through homology-directed repair (HDR). This requires a repair template with homology arms for both free DNA ends produced by the DSB. Consequently, CRISPR-Cas12a facilitates specific sequence integration when it is used alongside a donor template (Fig. 1 ). When the donor is a single-stranded DNA molecule, HDR is optimal when homology arms are around 80nt long [ 14 ], against 800 nt for double-stranded donor templates. To prevent new cleavage after the insertion, it is necessary to ensure that the crRNA is not able to bind the corrected locus. This can be achieved by ensuring that the PAM or part of the target sequence are deleted upon repair or by introducing synonymous mutations in the repair template [ 15 ]. Gene repair: HDR can also be harnessed in order to revert mutations, using a donor highly homologous to the targeted locus but carrying the wild-type allele instead of the mutation to be repaired (Fig. 1 ). Again, a usual method to prevent crRNA binding to the corrected sequence is to add synonymous mutations in the sequence complementary to the gRNA [ 15 ]. Fig. 1 Repair mechanisms of Cas12a-induced DSB. Double-strand breaks generated by Cas12a in target cells can be repaired by non-homologous end joining (NHEJ) or homology-directed repair (HDR). NHEJ sometimes produces indels at the repair site and can thus be harnessed for knock-out applications. HDR can be harnessed to reverse mutations or insert transgenes depending on the donor co-delivered with the CRISPR-Cas12a system
Knock-outs: In higher eukaryotes, non-dividing haploid cells and eukaryotic cells in the G1 phase of the cell cycle, the main DNA repair pathway is non-homologous end joining (NHEJ) [ 12 ], a relatively error-prone pathway. Thus, a sequence targeted by CRISPR-Cas12a can be cut repeatedly by the nuclease until the repair mechanisms introduce indels (Fig. 1 ). When these mutations occur in exons, they can induce frame-shifts or less frequently insert stop codons or disrupt essential amino acids or splicing sites [ 7 ]. In each case, the protein produced by the targeted gene is likely to be inactivated or may not

Regulation of the CRISPR-Cas12a system by methylation and demethylation of guide RNA. — Zhian Hu et al., 2023

Clustered regularly interspaced short palindromic repeats (CRISPR) – Cas technologies have revolutionized fields ranging from fundamental science to medical therapies. 1,2 However, challenges of spatial and temporal control of CRISPR-Cas need to be addressed, which reduces potential off-target editing and broadens medical applications. Exogenously inducible CRISPR-Cas tools have been first developed, 3 such as employing blue light 4,5 and small drug molecules 6–8 to precisely activate the CRISPR-Cas system to induce interest gene expression. To fully exert the potential of CRISPR in living cells, CRISPR tools need to be manipulated using crucial endogenous biomolecules for studying intracellular biomarkers and controlling the genome of specific cells such as cancer cells and stem cells. 9,10 For example, a microRNA-inducible CRISPR platform has been proposed for serving as a stem cell genome-regulation tool and sensing the microRNA. 9
Chemical modifications on gRNA can also efficiently regulate the performance of CRISPR-Cas. 8,11–13 Many chemical modifications on ribose sugar and the backbone have been investigated so far, including 2′-F, 14,15 universal bases (inosine and 5′ nitroindole), 16 locked nucleic acid (LNA), 17 phosphorothioates (PS), 18 etc . These chemical modifications could significantly reduce off-target editing and strengthen CRISPR-Cas system bioavailability in vivo . Recently, conditional control of chemical modifications on gRNA, such as light caged modifications 19–21 and small-ligand caged modifications, 8,22 has sprung up. For example, Deiters' group has achieved light-controllable gene editing in living cells and zebrafish through 6-NPOM-caged modifications on gRNA of Cas9. 13 These modifications can further spatiotemporally control CRSIPR in vivo for precise gene-editing and regulation. Despite the achievements, existing chemical modifications are commonly artificial rather than natural intracellular RNA modifications, which are scarcely related to cellular behaviours, such as epigenetic regulation. 10 In general, endogenous chemical modifications, such as methylation, are often associated with vital cellular processes. 23 By exploiting endogenous reversible chemical modifications to regulate the activity of CRISPR systems, we may be able to minimize their off-target effects while conferring them with cell

-type or cell-state specificity, and even apply them for biosensing and therapeutic applications. Therefore, it is promising to investigate endogenous chemical modifications on gRNA to control CRISPR-Cas. 12
The CRISPR-Cas effector is similar to the ribosome, working as ribonucleoprotein particles (RNPs). 24,25 Emerging evidence demonstrates that epigenetic modifications on the RNA component can precisely regulate the function and structure of RNPs, playing an essential role in almost every aspect of cellular regulation and growth. 26–28 Among these RNA epigenetic markers, N 6 -methyladenosine (m6A) and N 1 -methyladenosine (m1A) are two of the few RNA modifications whose reversible reaction pathway in cells has been fully elucidated. 29 m6A could be erased from RNA by ALKBH5 and FTO demethylases while m1A could be removed by ALKBH3 demethylase. 30–32 These demethylases maintain dynamic m6A and m1A expression in living cells and play vital roles in promoting cell proliferation and regulating RNA-protein interactions. 33 Furthermore, regulation of CRISPR activity by naturally occurring mRNA modifications has scarcely been systematically studied before. Therefore, we expect that methylation and demethylation of CRISPR gRNA may be an effective way to control the activity of the CRISPR system when combined with demethylases.
In this work, we found that the m6A or m1A modification in the 5′ handle of gRNA could significantly inhibit the cis - and trans -cleavage activity of CRISPR-Cas12a. Referring to the structure of the Cas12a effector, 34 methylation may hinder or disrupt the pseudoknot structure of gRNA, which prevents the formation of a stable complex between the Cas12a protein and gRNA, resulting in the loss of DNA cleavage capability. The gRNA structure could be restored by demethylases through erasing the methyl group. The m6A methylation-deactivated CRISPR-Cas12a nuclease can be robustly reactivated by ALKBH5 and FTO, while the m1A methylation-deactivated CRISPR-Cas12a can be reactivated by ALKBH3 demethylases. Using the trans -cleavage activity of Cas12a, we were able to successfully develop a reliable and

sensitive fluorescence read-out strategy for monitoring the activity of demethylases. Moreover, we successfully used the demethylase to control gene editing. In addition, we constructed a specific dCas12a transcriptional circuit that permits inducible protein expression in living cells by demethylase-mediated gRNA activation. Overall, we proposed a novel strategy for manipulating CRISPR-Cas12a activity by controlling chemical modifications of gRNA ( Scheme 1 ).
As a proof of concept, the effect of m6A modification on different regions of gRNA has been investigated in detail. As shown in Fig. 1A , the 5′ handle pseudoknot structure of gRNA (red box) is tightly folded and particularly sensitive to chemical modifications. 12 The remaining 20 nts of the gRNA (blue box) serve as the spacer that recognizes and invades the target dsDNA. Both regions of the gRNA are important for efficient DNA cleavage. Generally, the methyl group in m6A causes a steric clash in Watson–Crick pairing of RNA ( Fig. 1B ). 26,35 Therefore, we expect that this modification may destabilize the pseudoknot structure or the pairing between the spacer and target DNA. We constructed BC-gRNA ( Fig. 1C ) containing m6A at sites A 2 , A 8 , A 16, and A 18 . As for the spacer, BR-gRNA ( Fig. 1C ) with m6A at sites A 22 , A 27 , A 31, and A 36 was designed. In addition, a gRNA with eight m6A methylations (8(m6A)-gRNA) was designed to effectively switch-off CRISPR-Cas12a ( Fig. 1C ).
Generally, CRISPR-Cas12a holds two cleavage activities including target specific cleavage ( cis -cleavage) activity and non-specific nucleic acid cleavage ( trans -cleavage) activity. In this work, we conducted separate tests to assess the impact of methylated gRNAs on two discrete activities of CRISPR-Cas12a. First, the cis -cleavage activity of Cas12a to the dsDNA substrate was investigated. Gel electrophoresis revealed that dsDNA could be effectively cleaved by Cas12a in the presence of normal gRNA ( Fig. 1D ). BR-gRNA displayed a slight inhibitory

Programmable RNA detection with CRISPR-Cas12a. — Piyush Jain et al., 2023

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is an adaptive immune system encoded within prokaryotes that have evolved to counter invasion by foreign nucleic acids such as bacteriophages and plasmids 1 , 2 . Upon infection, the invading DNA sequences are captured and integrated into the host genome between an array of repeat sequences. The captured DNA sequences are called ‘spacers’ and they provide a genetic memory of prior infections 3 . For prokaryotic immunity, the CRISPR locus is transcribed and processed to generate multiple mature CRISPR RNAs (crRNA), each encoding a unique spacer sequence. Cas (CRISPR-associated) proteins are RNA-guided endonucleases that when complexed with the CRISPR RNAs (crRNAs) can enable the cleavage of nucleic acids that are complementary to the crRNA sequence.
There are several diverse naturally occurring CRISPR/Cas systems found in prokaryotes 4 , 5 . Among these, Cas12a is a class II, type V RNA-guided DNA endonuclease 6 . Since its discovery, it has been widely used for genome editing as well as molecular diagnostic applications 7 – 11 . Structural and biochemical studies have shown that Cas12a can catalyze the cleavage of DNA substrates 12 – 14 but there are no reports of targeted RNA cleavage by Cas12a. Recently there has been a report of a novel enzyme named Cas12a2 that sometimes co-occurs with Cas12a systems in bacteria and can use the Cas12a crRNA, but recognizes an RNA target instead of a double-stranded DNA 15 . A special characteristic of the type V Cas12 family of enzymes is their ability to initiate rapid and indiscriminate cleavage of any non-specific single-stranded DNA (ssDNA) molecules in their vicinity after target-specific recognition and cleavage 16 , 17 . This unique catalytic property, known as trans -cleavage has been harnessed to engineer CRISPR-based diagnostic tools that rely on the cleavage of FRET reporters upon target recognition 18 .
So far, Cas12a-based tools have been limited to the detection of DNA substrates, unless they are coupled with additional steps involving reverse transcription or strand displacement 19 – 21 . Recently, we discovered that CRISPR-Cas12a can tolerate DNA/RNA heter

oduplexes, but only when RNA is located at the non-target strand 22 . We and others utilized the system to detect RNA targets with Cas12a by simply creating a heteroduplex using a reverse transcription step without amplification 22 , 23 . A reverse-transcription step is inconvenient because it adds to the time, cost, error, and complexity of the assay. The other alternative is to use an RNA-targeting enzyme such as Cas12a2 15 , Cas12g 24 , or Cas13a-d 25 – 27 ; however, these systems can only detect RNA.
Cas12a orthologs are known to require a short protospacer adjacent motif (PAM) to be present on the target DNA to initiate recognition and cleavage 6 . It has been previously shown that DNA-cleaving enzymes such as Cas9 can be manipulated to also cleave RNA through the addition of a PAMmer sequence 28 . However, similar approaches have not yet been investigated with trans -cleaving Cas enzymes like Cas12, primarily because the PAM recognition mechanism is very different between Cas9 and Cas12 enzymes 29 . For instance, unlike Cas9, Cas12a can recognize and even trigger trans -cleavage activity using a PAM-less ssDNA. To date, there is no single CRISPR-Cas system identified that can innately tolerate both DNA and RNA substrates to trigger trans -cleavage.
In this report, we discovered that Cas12a can also tolerate RNA substrates at the PAM-distal end of the crRNA and initiate trans -cleavage activity. In essence, we have found that while the PAM-proximal seed region of the crRNA strictly tolerates DNA substrates, the PAM-distal end of the crRNA can tolerate both RNA and DNA substrates with multiple Cas12a orthologs. Thus, by merely supplying a short ssDNA or a PAM-containing dsDNA at the seed region of the crRNA we can detect RNA substrates at the 3’-end of the crRNA. We harnessed this unique property to develop a tool for RNA detection using Cas12a named S plit A ctivators for H ighly A ccessible R NA A nalysis (SAHARA).
We achieved reverse transcription (RTx)-free detection of picomolar levels of DNA as well as RNA without amplification using SA

HARA and applied it for detecting clinically-relevant targets including hepatitis C virus (HCV) RNA and microRNA-155 (miR-155). We showed that compared to conventional CRISPR-Cas12a, SAHARA has improved specificity and can be performed at room temperature. We demonstrate that its activity can be turned ON or OFF using the seed region binding DNA activator as a switch. We took advantage of this switch to perform multiplexed and simultaneous detection of different DNA and RNA targets. We also coupled SAHARA with Cas13b to perform multiplexed detection of different RNA targets. These key findings provided insights into the substrate requirements for the trans -cleavage activity of Cas12a, and we have utilized them to develop SAHARA, a valuable and versatile tool that can simultaneously detect both DNA and RNA substrates.
LbCas12a, AsCas12a, and ErCas12a are orthologs of Cas12a nucleases that are derived from the Lachnospiraceae bacterium ND2006, Acidaminococcus sp . BVL36, and Eubacterium rectale are simply referred to here as Lb, As, and Er, respectively 30 – 33 (Fig. S1). The mature crRNAs for each ortholog are 41–44 nt in length, containing ~19–21 nt of scaffold and the remaining ~20–24 nt of spacer 6 .
We wondered whether two different ssDNA targets, each binding to a different position of the same crRNA, can be used to initiate the trans -cleavage activity of Cas12a ( Fig. 1a , S2). To test the minimum length of activators tolerated by Cas12a, we designed several short ssDNA target activators of lengths ranging from 6–20 nt that were complementary to either the PAM-proximal (Pp) seed region or the PAM-distal (Pd) end of the crRNA ( Fig. 1b – d ).
We first performed in vitro trans -cleavage assays with individual truncated activators using three different orthologs of Cas12a. We found that the trans -cleavage activity is extremely sensitive to truncations of the ssDNA activators across the tested orthologs ( Fig. 1b – d ). Compared to the full-length 20-nt activator, the trans -cleavage activity

Programmable RNA detection with CRISPR-Cas12a — Santosh R. Rananaware et al., 2023

Introduction CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is an adaptive immune system encoded within prokaryotes that has evolved to counter invasion by foreign nucleic acids from bacteriophages and plasmids 1 , 2 . Upon infection, the invading DNA sequences are captured and integrated into the host genome between an array of repeat sequences. The captured DNA sequences are called ‘spacers’ and they provide a genetic memory of prior infections 3 . For prokaryotic immunity, the CRISPR locus is transcribed and processed to generate multiple mature CRISPR RNAs (crRNA), each encoding a unique spacer sequence. The crRNA forms a complex with an RNA-guided endonuclease, Cas (CRISPR-associated) protein. In the presence of nucleic acids complementary to the crRNA sequence, the CRISPR/Cas system enables the cleavage of nucleic acids. There are several diverse naturally occurring CRISPR/Cas systems found in prokaryotes 4 , 5 . Among these, Cas12a is a class 2, type V RNA-guided DNA endonuclease 6 . Since its discovery, it has been widely used for genome editing as well as molecular diagnostic applications 7 , 8 , 9 , 10 , 11 . Structural and biochemical studies have shown that Cas12a can catalyze the cleavage of DNA substrates 12 , 13 , 14 but there are no reports of targeted RNA cleavage by Cas12a, without pre-processing steps such as reverse transcription. Recently there has been a report of a novel enzyme named Cas12a2 that sometimes co-occurs with Cas12a systems in bacteria and can use the Cas12a crRNA, but recognizes an RNA target instead of a double-stranded DNA 15 . A special characteristic of the type V Cas12 family of enzymes is their ability to initiate rapid and indiscriminate cleavage of any non-specific single-stranded DNA (ssDNA) molecules in their vicinity after target-specific recognition and cleavage 16 , 17 . This unique catalytic property, known as trans- cleavage has been harnessed to engineer CRISPR-based diagnostic tools that rely on the cleavage of FRET reporters upon target recognition 18 . So far, Cas12a-based tools have been limited to the detection of DNA substrates, unless they are coupled with additional steps involving reverse transcription or strand displacement

19 , 20 , 21 . Previously we have shown that CRISPR-Cas12a can tolerate DNA/RNA heteroduplexes, but only when RNA is located at the non-target strand 22 . This can be used to detect RNA targets with Cas12a by simply creating a heteroduplex using a reverse transcription step without amplification 22 , 23 . A reverse transcription step is inconvenient because it adds to the time, cost, error, and complexity of the assay. The other alternative is to use an RNA-targeting enzyme such as Cas12a2 15 , Cas12g 24 , or Cas13a-d 25 , 26 , 27 ; however, these systems can only detect RNA. Cas12a orthologs are known to require a short protospacer adjacent motif (PAM) to be present on the target DNA to initiate recognition and cleavage 6 . It has been previously shown that DNA-cleaving enzymes such as Cas9 can be manipulated to also cleave RNA through the addition of a PAMmer sequence, a PAM-containing oligonucleotide, which is annealed to the target ssRNA to initiate cleavage 28 . However, similar approaches have not yet been investigated with trans- cleaving Cas enzymes like Cas12, primarily because the PAM recognition mechanism is different between Cas9 and Cas12 enzymes 29 . For instance, unlike Cas9, Cas12a can recognize and even trigger trans- cleavage activity using a PAM-less ssDNA. To date, there is no single CRISPR-Cas system identified that can innately tolerate both DNA and RNA substrates to trigger trans- cleavage. In this work, we show that Cas12a can tolerate RNA substrates at the PAM-distal end of the crRNA and initiate trans- cleavage activity. While the PAM-proximal seed region of the crRNA strictly tolerates DNA substrates, the PAM-distal end of the crRNA can tolerate both RNA and DNA substrates with multiple Cas12a orthologs. Thus, by merely supplying a short ssDNA or a PAM-containing dsDNA at the seed region of the crRNA we can detect RNA substrates at the 3’-end of the crRNA. This specific property is the basis of our method for RNA detection using Cas12a named S plit A ctivators for H ighly A ccess

ible R NA A nalysis (SAHARA). SAHARA achieves reverse transcription (RTx)-free detection of picomolar levels of DNA as well as RNA without amplification and can detect clinically relevant targets including hepatitis C virus (HCV) RNA and microRNA-155 (miR-155). We show that SAHARA works robustly at room temperature and has improved specificity for point mutation detection compared to conventional CRISPR/Cas12a systems.We demonstrate that its activity requires the seed region DNA which allows for multiplexed assays. We use this switch capability to simultaneously detect different DNA and RNA targets. We also show that SAHARA works with Cas13b to perform multiplexed detection of different RNA targets. These key findings provide insight into the substrate requirements for the trans- cleavage activity of Cas12a, which are essential for SAHARA, a valuable and versatile tool that can simultaneously detect both DNA and RNA substrates.
Results Cas12a orthologs tolerate split ssDNA activators for trans- cleavage activity LbCas12a, AsCas12a, and ErCas12a are orthologs of Cas12a nucleases that are derived from Lachnospiraceae bacterium ND2006, Acidaminococcus sp. BVL36, and Eubacterium rectale and are simply referred to here as Lb, As, and Er, respectively 30 , 31 , 32 , 33 (Fig. S 1 ). The mature crRNAs for each ortholog are 41–44 nt in length, containing ~19–21 nt of scaffold and the remaining ~20-24 nt of spacer 6 . We wondered whether two different ssDNA targets, each binding to a different position of the same crRNA, can be used to initiate the trans- cleavage activity of Cas12a (Fig. 1a , S 2 ). To test the minimum length of activators tolerated by Cas12a, we designed several short ssDNA target activators of lengths ranging from 6-20 nt that were complementary to either the PAM-proximal (Pp) seed region or the PAM-distal (Pd) end of the crRNA (Fig. 1b–d , S 3 ). Fig. 1: Cas12a orthologs tolerate short ssDNA activators (6-12 nt) when added in combination. a Schematic representation of