Engineered CRISPR/Cas9 enzymes improve discrimination by slowing DNA cleavage to allow release of off-target DNA

Liu, Mu‐Sen; Gong, Shanzhong; Yu, Helen‐Hong; Jung, Kyungseok; Johnson, Kenneth A.; Taylor, David W.

doi:10.1038/s41467-020-17411-1

Cited by 66 publications

(67 citation statements)

References 34 publications

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“…In this respect, it is worth noting that DNA binding-induced conformational changes occur at remarkably slower rates than what is possible to simulate using classical MD. 36 , 37 Yet, our data show a good coverage of the conformational landscape ( Figures 3 b and S7 ), implying that the PCA well-represented the large-scale dynamics of the system. It is also notable that in the DNA-bound states, the conformational space explored by the protein is slightly restricted as a result of global stabilization due to the binding of the DNA (FnCas12a) and a complete NTS (FnCas12a′).…”

Section: Resultssupporting

confidence: 54%

Molecular Dynamics Reveals a DNA-Induced Dynamic Switch Triggering Activation of CRISPR-Cas12a

Saha

Arantes

Hsu

et al. 2020

J. Chem. Inf. Model.

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CRISPR-Cas12a is a genome-editing system, recently also harnessed for nucleic acid detection, which is promising for the diagnosis of the SARS-CoV-2 coronavirus through the DETECTR technology. Here, a collective ensemble of multimicrosecond molecular dynamics characterizes the key dynamic determinants allowing nucleic acid processing in CRISPR-Cas12a. We show that DNA binding induces a switch in the conformational dynamics of Cas12a, which results in the activation of the peripheral REC2 and Nuc domains to enable cleavage of nucleic acids. The simulations reveal that large-amplitude motions of the Nuc domain could favor the conformational activation of the system toward DNA cleavages. In this process, the REC lobe plays a critical role. Accordingly, the joint dynamics of REC and Nuc shows the tendency to prime the conformational transition of the DNA target strand toward the catalytic site. Most notably, the highly coupled dynamics of the REC2 region and Nuc domain suggests that REC2 could act as a regulator of the Nuc function, similar to what was observed previously for the HNH domain in the CRISPR-associated nuclease Cas9. These mutual domain dynamics could be critical for the nonspecific binding of DNA and thereby for the underlying mechanistic functioning of the DETECTR technology. Considering that REC is a key determinant in the system’s specificity, our findings provide a rational basis for future biophysical studies aimed at characterizing its function in CRISPR-Cas12a. Overall, our outcomes advance our mechanistic understanding of CRISPR-Cas12a and provide grounds for novel engineering efforts to improve genome editing and viral detection.

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Section: Resultssupporting

confidence: 54%

Molecular Dynamics Reveals a DNA-Induced Dynamic Switch Triggering Activation of CRISPR-Cas12a

Saha

Arantes

Hsu

et al. 2020

J. Chem. Inf. Model.

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“…Among the HF Cas9s, HiFi Cas9 had cleavage rates that were comparable to WT SpCas9. In contrast, SpCas9 HF1 and HypaCas9 cleaved pTarget PS4 ∼36- and ∼12-fold slower than SpCas9, respectively (Figure 1B , C , Supplementary Figure S2B ), similar to previously reported cleavage defects for these two HF Cas9 variants ( 52 ). However, cleavage rates for SpCas9 HF1 and HypaCas9 were comparable to SpCas9 for pTarget EMX1 (Figure 1C , Supplementary Figure S2A, B ), indicating that cleavage defects for HF Cas9 variants may vary based on target sequence.…”

Section: Resultssupporting

confidence: 89%

“…However, many previous studies investigated individual Cas9 variants separately, focusing on target binding and/or DSB formation by Cas9. Our in vitro library cleavage assay has enabled a comparative study of the cleavage specificity of Cas9 variants, revealing cleavage defects that have previously remained undetected ( 6 , 14 , 15 , 38 , 40 , 52 ). We find that engineered SpCas9 variants display higher specificity than wild-type SpCas9 in a target-dependent manner, although prolonged exposure reduces this specificity.…”

Section: Discussionmentioning

confidence: 99%

Systematicin vitrospecificity profiling reveals nicking defects in natural and engineered CRISPR–Cas9 variants

Murugan

Suresh

Seetharam

et al. 2021

Nucleic Acids Research

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Cas9 is an RNA-guided endonuclease in the bacterial CRISPR–Cas immune system and a popular tool for genome editing. The commonly used Streptococcus pyogenes Cas9 (SpCas9) is relatively non-specific and prone to off-target genome editing. Other Cas9 orthologs and engineered variants of SpCas9 have been reported to be more specific. However, previous studies have focused on specificity of double-strand break (DSB) or indel formation, potentially overlooking alternative cleavage activities of these Cas9 variants. In this study, we employed in vitro cleavage assays of target libraries coupled with high-throughput sequencing to systematically compare cleavage activities and specificities of two natural Cas9 variants (SpCas9 and Staphylococcus aureus Cas9) and three engineered SpCas9 variants (SpCas9 HF1, HypaCas9 and HiFi Cas9). We observed that all Cas9s tested could cleave target sequences with up to five mismatches. However, the rate of cleavage of both on-target and off-target sequences varied based on target sequence and Cas9 variant. In addition, SaCas9 and engineered SpCas9 variants nick targets with multiple mismatches but have a defect in generating a DSB, while SpCas9 creates DSBs at these targets. Overall, these differences in cleavage rates and DSB formation may contribute to varied specificities observed in genome editing studies.

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“…Yet, engineering efforts designed around Spy Cas9 binding have achieved greater on-target efficacy and specificity (39). More broadly, off-target detection methods have demonstrated substantial time-and concentration-dependent off-target activity (42,43), and kinetic partitioning has been found to underpin enhanced specificity of engineered Cas9 derivatives (44). For this reason, protein engineering efforts are unlikely to offer a single solution for experiments that operate over different time scales with different tolerance for off-target effects, and more advanced biophysical models for Cas9 activity remain a top priority.…”

Section: Discussionmentioning

confidence: 99%

Quantification of Cas9 binding and cleavage across diverse guide sequences maps landscapes of target engagement

et al. 2021

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The RNA-guided nuclease Cas9 has unlocked powerful methods for perturbing both the genome through targeted DNA cleavage and the regulome through targeted DNA binding, but limited biochemical data have hampered efforts to quantitatively model sequence perturbation of target binding and cleavage across diverse guide sequences. We present scalable, sequencing-based platforms for high-throughput filter binding and cleavage and then perform 62,444 quantitative binding and cleavage assays on 35,047 on- and off-target DNA sequences across 90 Cas9 ribonucleoproteins (RNPs) loaded with distinct guide RNAs. We observe that binding and cleavage efficacy, as well as specificity, vary substantially across RNPs; canonically studied guides often have atypically high specificity; sequence context surrounding the target modulates Cas9 on-rate; and Cas9 RNPs may sequester targets in nonproductive states that contribute to “proofreading” capability. Lastly, we distill our findings into an interpretable biophysical model that predicts changes in binding and cleavage for diverse target sequence perturbations.

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Engineered CRISPR/Cas9 enzymes improve discrimination by slowing DNA cleavage to allow release of off-target DNA

Cited by 66 publications

References 34 publications

Molecular Dynamics Reveals a DNA-Induced Dynamic Switch Triggering Activation of CRISPR-Cas12a

Molecular Dynamics Reveals a DNA-Induced Dynamic Switch Triggering Activation of CRISPR-Cas12a

Systematicin vitrospecificity profiling reveals nicking defects in natural and engineered CRISPR–Cas9 variants

Quantification of Cas9 binding and cleavage across diverse guide sequences maps landscapes of target engagement

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