Highly Parallel Profiling of Cas9 Variant Specificity

Schmid-Burgk, Jonathan L.; Gao, Linyi; Li, David; Gardner, Zachary J G; Strecker, Jonathan; Lash, Blake; Zhang, Feng

doi:10.1016/j.molcel.2020.02.023

Cited by 145 publications

(173 citation statements)

References 32 publications

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“… 17 − 21 These studies provided predictive insights into the dynamic behavior of Cas9, many of which have been corroborated by single-molecule FRET experiments 23 and cryoEM, 24 providing also a framework for rational design of Cas9 for improved genome editing. 25 , 26 …”

Section: Introductionmentioning

confidence: 99%

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: Introductionmentioning

confidence: 99%

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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“…Our analysis revealed that the high specificity of SaCas9 and HF Cas9 variants observed in previous studies may stem from the lack of detection of nicking by Cas9. Most studies test for DSB at target and off-target sites and/or indel formation Huston et al, 2019;Kleinstiver et al, 2016;Schmid-Burgk et al, 2020;Vakulskas et al, 2018;Zhang et al, 2020). While SpCas9 linearized most target sequences with multiple mismatches as previously observed, SaCas9 and HF Cas9 variants often only nicked these sequences.…”

Section: Discussionmentioning

confidence: 97%

“…Recent in vitro and in vivo studies profiled kinetic properties and genome editing activities of natural and high-fidelity Cas9 variants (Jones et al, 2019;Schmid-Burgk et al, 2020). In these studies, SpCas9 had high mismatch-tolerance and in turn, more off-target cleavage activity whereas HF Cas9 variants had reduced off-target cleavage activity.…”

Section: Discussionmentioning

confidence: 99%

“…DNA accessibility can also vary depending on cellular processes which may change the outcome and detection of potential Cas9 off-target editing events. These methods also rely on DSBs in the DNA generated by Cas9 or post-cleavage DNA repair and indel formation which can vary among cell types and experiments (Schmid-Burgk et al, 2020;Tsai et al, 2017Tsai et al, , 2015.…”

Section: Introductionmentioning

confidence: 99%

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High-throughput in vitro specificity profiling of natural and high-fidelity CRISPR-Cas9 variants

Murugan

Seetharam

Severin

et al. 2020

Preprint

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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 most commonly used Cas9 variant, 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 than wildtype (WT) SpCas9. However, systematic comparisons of the cleavage activities of these Cas9 variants have not been reported. In this study, we employed our high -throughput in vitro cleavage assay to 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 were able to cleave target sequences with up to five mismatches. However, the rate of cleavage of both on-target and offtarget sequences varied based on the target sequence and Cas9 variant. For targets with multiple mismatches, SaCas9 and engineered SpCas9 variants are more prone to nicking, while WT SpCas9 creates double-strand breaks (DSB). These differences in cleavage rates and DSB formation may account for the varied specificities observed in genome editing studies. Our analysis reveals mismatch position-dependent, off-target nicking activity of Cas9 variants which have been underreported in previous in vivo studies.

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“…However, the off-target effect of Cas9 on monkey genomes and other species is still inevitable, which limits its’ widespread applications(Zhang et al, 2015). Recent studies have demonstrated that CRISPR-Cas9 off-target effects can be reduced by increasing Cas9’s specificity including the use of high-fidelity Cas9 variants(Kleinstiver et al, 2016,Schmid-Burgk et al, 2020), or by reducing the generation of DSBs on off-target sites including the use of truncated sgRNAs or paired sgRNA/Cas9-D10A nickases(Bin et al, 2014,F Ann et al, 2013,Gopalappa et al, 2018). However, some of these methods often achieve lower off-target effects at the expense of reducing on-target efficiency.…”

Section: Introductionmentioning

confidence: 99%

Mutating PINK1 gene by paired truncated sgRNA/Cas9-D10A in Cynomolgus Monkey

Chen

Kang

Yang

et al. 2020

Preprint

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Mutations of PINK1 cause early-onset Parkinson's disease (PD) with selective neurodegeneration in humans. However, current PINK1 knockout mouse and pig models are unable to recapitulate the typical neurodegenerative phenotypes observed in PD patients, indicating that it is essential to generate PINK1 disease models in non-human primates (NHPs) that are highly close to humans, to investigate the unique function of PINK1 in primate brains. Paired single guide RNA (sgRNA)/Cas9-D10A nickases and truncated sgRNA/Cas9, both enabling the reduction of the off-target effect without apparently compromising the on-target editing, are two optimized strategies in applying the CRISPR/Cas9 system to establish disease animal models. Here, we combined the two strategies and injected Cas9-D10A mRNA and two truncated sgRNAs into one-cell-stage cynomolgus zygotes to target PINK1 gene. We show precise and efficient gene editing of the target site in the three new born cynomolgus monkeys. The frame shift mutations of PINK1 in mutant fibroblasts leaded to the reduction of the mRNA, However, western-blot and immunofluorescence staining confirmed the PINK1 protein levels were comparable to that in the wild-type fibroblasts. We further reprogramed mutant fibroblast into induced pluripotent stem cells (iPSCs) and they show similar ability of differentiation into dopamine (DA) neurons. Taken together, our results showed that coinjection of Cas9-D10A nickase mRNA and sgRNA into one-cell-stage cynomolgus embryos enable the generation of human disease models in NHPs and the target editing by paired truncated sgRNA/Cas9-D10A in PINK1 gene exon 2 did not impact the protein expression.

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Highly Parallel Profiling of Cas9 Variant Specificity

Cited by 145 publications

References 32 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

High-throughput in vitro specificity profiling of natural and high-fidelity CRISPR-Cas9 variants

Mutating PINK1 gene by paired truncated sgRNA/Cas9-D10A in Cynomolgus Monkey

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