The bridge helix of Cas12a imparts selectivity in <i>cis</i>‐DNA cleavage and regulates <i>trans</i>‐DNA cleavage

Parameshwaran, Hari Priya; Babu, Kesavan; Tran, Christine; Guan, Kevin; Allen, Aleique; Kathiresan, Venkatesan; Qin, Peter Z.; Rajan, Rakhi

doi:10.1002/1873-3468.14051

Cited by 11 publications

(18 citation statements)

References 70 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Distortion of the PAM double-stranded DNA (dsDNA) leads to strand separation and R-loop formation within the recognition lobe, starting with the pre-structured 5′ end of the crRNA spacer (the ‘seed’) 16 , 20 , 22 , 25 . As R-loop formation progresses, conformational checkpoints need to be passed so the catalytic pocket is made available to bind any ssDNA 14 , 20 , 26 , 27 (Extended Data Fig. 1 ).…”

Section: Mainmentioning

confidence: 99%

CRISPR–Cas12a-mediated DNA clamping triggers target-strand cleavage

et al. 2022

View full text Add to dashboard Cite

Clustered regularly interspaced short palindromic repeats (CRISPR)–Cas12a is widely used for genome editing and diagnostics, so it is important to understand how RNA-guided DNA recognition activates the cleavage of the target strand (TS) following non-target-strand (NTS) cleavage. Here we used single-molecule magnetic tweezers, gel-based assays and nanopore sequencing to explore DNA unwinding and cleavage. In addition to dynamic and heterogenous R-loop formation, we also directly observed transient double-stranded DNA unwinding downstream of the 20-bp heteroduplex and, following NTS cleavage, formation of a hyperstable ‘clamped’ Cas12a–DNA intermediate necessary for TS cleavage. Annealing of a 4-nucleotide 3′ CRISPR RNA overhang to the unwound TS downstream of the heteroduplex inhibited clamping and slowed TS cleavage by ~16-fold. Alanine substitution of a conserved aromatic amino acid in the REC2 subdomain that normally caps the R-loop relieved this inhibition but favoured stabilisation of unwound states, suggesting that the REC2 subdomain regulates access of the 3′ CRISPR RNA to downstream DNA.

show abstract

Section: Mainmentioning

confidence: 99%

CRISPR–Cas12a-mediated DNA clamping triggers target-strand cleavage

et al. 2022

View full text Add to dashboard Cite

show abstract

“…However, this ability provided by an intact BH to tolerate DNA mismatches is a pitfall in gene editing applications, which require high specificity in DNA cleavage. Our data emphasizes that modulating the BH can be a promising strategy for the enhancement of specificity of Cas9 or other type-V Cas effectors since the BH is conserved among these proteins. , …”

Section: Discussionmentioning

confidence: 74%

Coordinated Actions of Cas9 HNH and RuvC Nuclease Domains Are Regulated by the Bridge Helix and the Target DNA Sequence

et al. 2021

Self Cite

View full text Add to dashboard Cite

CRISPR-Cas systems are RNA-guided nucleases that provide adaptive immune protection in bacteria and archaea against intruding genomic materials. Cas9, a type-II CRISPR effector protein, is widely used for gene editing applications since a single guide RNA can direct Cas9 to cleave specific genomic targets. The conformational changes associated with RNA/DNA binding are being modulated to develop Cas9 variants with reduced off-target cleavage. Previously, we showed that proline substitutions in the arginine-rich bridge helix (BH) of Streptococcus pyogenes Cas9 (SpyCas9-L64P-K65P, SpyCas9 2Pro ) improve target DNA cleavage selectivity. In this study, we establish that kinetic analysis of the cleavage of supercoiled plasmid substrates provides a facile means to analyze the use of two parallel routes for DNA linearization by SpyCas9: (i) nicking by HNH followed by RuvC cleavage (the TS (target strand) pathway) and (ii) nicking by RuvC followed by HNH cleavage (the NTS (nontarget strand) pathway). BH substitutions and DNA mismatches alter the individual rate constants, resulting in changes in the relative use of the two pathways and the production of nicked and linear species within a given pathway. The results reveal coordinated actions between HNH and RuvC to linearize DNA, which is modulated by the integrity of the BH and the position of the mismatch in the substrate, with each condition producing distinct conformational energy landscapes as observed by molecular dynamics simulations. Overall, our results indicate that BH interactions with RNA/DNA enable target DNA discrimination through the differential use of the parallel sequential pathways driven by HNH/RuvC coordination.

show abstract

“…As Cas12a utilizes a single RuvC domain to cleave sequentially the two strands of dsDNA and then catalyze trans -ssDNA cutting (5, 14, 18, 19), we tried to understand how the dual activities of the RuvC domain are regulated by kinetic analyses of LbCas12a proteins with mutations in its bridge helix domain. From four variants that were tested (mut1-4), we chose mut2 LbCas12a (a point mutation of W890A) for further study due to its relatively robust cis -activity and minimal trans -activity (Figure 1C, Supplementary Figure S1C-F).…”

Section: Resultsmentioning

confidence: 99%

Improved Genome Editing by an Engineered CRISPR-Cas12a

Chen

Shi

et al. 2022

Preprint

View full text Add to dashboard Cite

CRISPR-Cas12a is an RNA-guided, programmable genome editing enzyme found within bacterial adaptive immune pathways. Unlike CRISPR-Cas9, Cas12a uses only a single catalytic site to both cleave target double-stranded DNA (dsDNA) (cis-activity) and indiscriminately degrade single-stranded DNA (ssDNA) (trans-activity). To investigate how the relative potency of cis- versus trans-DNase activity affects Cas12a-mediated genome editing, we first used structure-guided engineering to generate variants of Lachnospiraceae bacterium Cas12a (LbCas12a) that selectively disrupt trans-activity. The resulting engineered mutant with the biggest differential between cis- and trans-DNase activity in vitro showed minimal genome editing activity in human cells, motivating a second set of experiments using directed evolution to generate additional mutants with robust genome editing activity. Notably, these engineered and evolved mutants had enhanced ability to induce homology-directed repair (HDR) editing by 2-18-fold depending on the genomic locus. Finally, we found that a site-specific reversion mutation produced improved Cas12a (iCas12a) variants with superior genome editing efficiency at genomic sites that are difficult to edit using wild-type Cas12a. This strategy of coupled rational engineering and directed evolution establishes a pipeline for creating improved genome editing tools by combining structural insights with randomization and selection. The availability of experimental and predicted structures of other CRISPR-Cas enzymes will enable this strategy to be applied to improve the efficacy of other genome editing proteins.

show abstract

The bridge helix of Cas12a imparts selectivity in cis‐DNA cleavage and regulates trans‐DNA cleavage

Cited by 11 publications

References 70 publications

CRISPR–Cas12a-mediated DNA clamping triggers target-strand cleavage

CRISPR–Cas12a-mediated DNA clamping triggers target-strand cleavage

Coordinated Actions of Cas9 HNH and RuvC Nuclease Domains Are Regulated by the Bridge Helix and the Target DNA Sequence

Improved Genome Editing by an Engineered CRISPR-Cas12a

Contact Info

Product

Resources

About