DNA unwinding is the primary determinant of CRISPR-Cas9 activity

Gong, Shanzhong; Yu, Helen; Johnson, Kenneth A.; Taylor, David W.

doi:10.1101/205823

Cited by 29 publications

(54 citation statements)

References 35 publications

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“…We found a strong agreement between our off-target cutting level and equilibrium dCas9 binding to the same mismatch positions/nucleotides, but no association with dCas9 binding rates for these same mismatched guides ( Figures 3C and 3D) (Boyle et al, 2017). This suggests that dissociation before cutting may be common at off-target sites in vivo, consistent with results observed in vitro and in human cell lines (Farasat and Salis, 2016;Gong et al, 2018;Ma et al, 2016a).…”

Section: (Legend Continued On Next Page)supporting

confidence: 86%

Functional Genetic Variants Revealed by Massively Parallel Precise Genome Editing

Sharon

Chen

Khosla

et al. 2018

Cell

186

216

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Section: (Legend Continued On Next Page)supporting

confidence: 86%

Functional Genetic Variants Revealed by Massively Parallel Precise Genome Editing

Sharon

Chen

Khosla

et al. 2018

Cell

186

216

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“…This would increase the time Cas9 has to equilibrate its binding and could result in preference of on-over off-target cleavage 37 . In accordance with this model, a recent study 38 showed that R-loop formation is the rate-limiting step, which reduces the DNA dissociation rate of Cas9. Replacing R63 and R66 increased the dissociation constant and/or decreased the cleavage rate of Cas9 by possibly destabilizing the R-loop.…”

Section: Discussionsupporting

confidence: 56%

Bridge helix arginines play a critical role in Cas9 sensitivity to mismatches

et al. 2020

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C as9 is an RNA-guided endonuclease that specifically binds and cleaves DNA with complementarity to the spacer sequence of CRISPR RNA (crRNA) located upstream of a protospacer adjacent motif (PAM) 1,2. Sufficient base pairing of the crRNA spacer of either dual-RNA (trans-activating crRNA (tracrRNA) in complex with crRNA) or single-guide RNA (sgRNA, tracrRNA fused to crRNA) with the target DNA strand leads to displacement of the nontarget strand and R-loop formation 1-5. On completion of this process, Cas9 endonuclease domains, HNH and RuvC, cleave the target and nontarget strand, respectively, resulting in a double-strand DNA break 1,2. Due to its programmability, CRISPR-Cas9 was adapted for a wide variety of genome engineering approaches 6-9. However, a main concern for the application of the CRISPR-Cas9 technology is off-target cleavage that occurs when Cas9 cleaves mismatched targets; that is, targets with imperfect complementarity to the dual-RNA/sgRNA spacer. It is therefore crucial to identify regions and amino acids that determine Cas9 specificity. Streptococcus pyogenes Cas9 (SpCas9) requires a seed sequence of 10-12 base pairs (bp) adjacent to the PAM for successful cleavage 2. The REC3 domain senses PAM-distal mismatches and controls the transition of the HNH domain into an active conformation, which serves as a mechanism to block the cleavage in the presence of mismatches 10,11. Alongside other strategies to reduce off-target cleavage 12 , both structure-guided and random mutagenesis have been used to generate enhanced specificity Cas9 variants 10,13-19. Three of the improved variants 10,15,17 contain mutations that increase the energetic barrier for transition of the HNH domain to the cleavage-competent state 10. eSpCas9 (ref. 17) and SpCas9-HF1 (ref. 15) require one additional base pair between crRNA and target DNA for stable binding, which makes target unwinding more sensitive to mismatches and slows cleavage in the presence of mismatches 20. It was shown that mutation of two residues in the loop region of the bridge helix impairs target cleavage and improves specificity 21. The conformational checkpoint through the HNH domain before cleavage and the seed sequence are the only known specificity determinants for Cas9. In addition, stable R-loop formation is an essential prerequisite for cleavage 5 , but residues involved in this process have not been identified. Therefore, we investigated whether other Cas9 domains are involved in mismatch sensing and R-loop stabilization focusing on the PAM-adjacent region of the target. We show that arginines of the Cas9 bridge helix influence guide RNA binding, and target DNA binding and cleavage. We identify two groups of arginines with opposite effects on Cas9 mismatch sensitivity. We demonstrate that R63, R66 and R70 stabilize the R-loop in the presence of PAM-adjacent mismatches thus reducing the Cas9 specificity. Additionally, we observe that Q768 mediates Cas9 insensitivity to a mismatch in target position 15 and is involved in sensitivity to PAM-distal mism...

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“…Once the R-loop is fully formed, the nuclease is activated and cleaves the target and non-target DNA strands to generate a DNA break 5 . R-loop extension is rate-limiting for cleavage by Cas12a and Cas9 in vitro [6][7][8][9] , providing a mechanistic explanation for Cas12a's observed higher specificity in vivo 8,[10][11][12][13][14][15] .…”

Section: Introductionmentioning

confidence: 88%

Inhibition of CRISPR-Cas12a DNA Targeting by Nucleosomes and Chromatin

Strohkendl

Saifuddin

Gibson

et al. 2020

Preprint

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Genome engineering nucleases, including CRISPR-Cas12a, must access chromatinized DNA. Here, we investigate how Acidaminococcus sp. Cas12a cleaves DNA within human nucleosomes and phase-condensed nucleosome arrays. Using quantitative kinetics approaches, we show that dynamic nucleosome unwrapping regulates DNA target accessibility to Cas12a. Nucleosome unwrapping determines the extent to which both steps of Cas12a binding-PAM recognition and R-loop formation-are inhibited by the nucleosome. Nucleosomes inhibit Cas12a binding even beyond the canonical core particle. Relaxing DNA wrapping within the nucleosome by reducing DNA bendability, adding histone modifications, or introducing a target-proximal nuclease-inactive Cas9 enhances DNA cleavage rates over 10-fold. Surprisingly, Cas12a readily cleaves DNA linking nucleosomes within chromatin-like phase separated nucleosome arrays-with DNA targeting reduced only ~4-fold. This work provides a mechanism for the observation that on-target cleavage within nucleosomes occurs less often than off-target cleavage within nucleosome-depleted regions of cells. We conclude that nucleosome wrapping restricts accessibility to CRISPR-Cas nucleases and anticipate that increasing nucleosome breathing dynamics will improve DNA binding and cleavage in eukaryotic cells.

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DNA unwinding is the primary determinant of CRISPR-Cas9 activity

Cited by 29 publications

References 35 publications

Functional Genetic Variants Revealed by Massively Parallel Precise Genome Editing

Functional Genetic Variants Revealed by Massively Parallel Precise Genome Editing

Bridge helix arginines play a critical role in Cas9 sensitivity to mismatches

Inhibition of CRISPR-Cas12a DNA Targeting by Nucleosomes and Chromatin

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