DNA stretching induces Cas9 off-target activity

Newton, Matthew D.; Taylor, Benjamin; Driessen, Rosalie P.C.; Roos, Leonie; Cvetešić, Nevena; Allyjaun, Shenaz; Lenhard, Boris; Cuomo, Maria Emanuela; Rueda, David

doi:10.1038/s41594-019-0188-z

Cited by 127 publications

(127 citation statements)

References 59 publications

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“…A variety of in vitro bulk assays, single‐molecule, computational, and crystallography approaches are being used to understand the mechanism of binding and cleaving of targets by the Cas9 complex . Different kinds of methods are used to detect and quantify off‐target effects caused by CRISPR/Cas9 and other CRISPR/Cas systems, and are described below ( Table 1 ).…”

Section: Methods/techniques To Detect Off‐target Effectsmentioning

confidence: 99%

“…DNA bubbles are involved in inducing binding and cleavage of off‐target sequence at these sites. The partly unwound or melted DNA helps in recruiting Cas9 for binding and cleaving formerly hidden cryptic off‐target sites . The mechanism of off‐target editing by the CRISPR/Cas system is illustrated in Figure .…”

Section: Mechanism Of Off‐target Effects By Crispr/cas Systemmentioning

confidence: 99%

“…A variety of experimental systems are widely being used to investigate the off‐target effects of sgRNAs and they show different outcomes regarding the extent of off‐targets . Many algorithms have been applied for the prediction of off‐target effects and many Cas9 proteins with high specificity have successfully been produced for minimizing Cas9 promiscuity . However, sequencing‐based approaches have been widely used to experimentally validate a variety of Cas9 off‐targets that are not fully explained by such algorithms, raising an interesting question about mechanisms of binding of Cas9 complexes and cleavage of off‐targets .…”

Section: Algorithms/tools For Sgrna Target Finding and Evaluation Anmentioning

confidence: 99%

“…Many algorithms have been applied for the prediction of off‐target effects and many Cas9 proteins with high specificity have successfully been produced for minimizing Cas9 promiscuity . However, sequencing‐based approaches have been widely used to experimentally validate a variety of Cas9 off‐targets that are not fully explained by such algorithms, raising an interesting question about mechanisms of binding of Cas9 complexes and cleavage of off‐targets . Many tools have been developed and widely used on the basis of mismatch information, including the number and location of the mismatches, finding and evaluating the potential off‐target sites.…”

Section: Algorithms/tools For Sgrna Target Finding and Evaluation Anmentioning

confidence: 99%

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CRISPR/Cas Systems in Genome Editing: Methodologies and Tools for sgRNA Design, Off‐Target Evaluation, and Strategies to Mitigate Off‐Target Effects

et al. 2020

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Life sciences have been revolutionized by genome editing (GE) tools, including zinc finger nucleases, transcription activator‐Like effector nucleases, and CRISPR (clustered regulatory interspaced short palindromic repeats)/Cas (CRISPR‐associated) systems, which make the targeted modification of genomic DNA of all organisms possible. CRISPR/Cas systems are being widely used because of their accuracy, efficiency, and cost‐effectiveness. Various classes of CRISPR/Cas systems have been developed, but their extensive use may be hindered by off‐target effects. Efforts are being made to reduce the off‐target effects of CRISPR/Cas9 by generating various CRISPR/Cas systems with high fidelity and accuracy. Several approaches have been applied to detect and evaluate the off‐target effects. Here, the current GE tools, the off‐target effects generated by GE technology, types of off‐target effects, mechanisms of off‐target effects, major concerns, and outcomes of off‐target effects in plants and animals are summarized. The methods to detect off‐target effects, tools for single‐guide RNA (sgRNA) design, evaluation and prediction of off‐target effects, and strategies to increase the on‐target efficiency and mitigate the off‐target impact on intended genome‐editing outcomes are summarized.

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Section: Methods/techniques To Detect Off‐target Effectsmentioning

confidence: 99%

Section: Mechanism Of Off‐target Effects By Crispr/cas Systemmentioning

confidence: 99%

Section: Algorithms/tools For Sgrna Target Finding and Evaluation Anmentioning

confidence: 99%

Section: Algorithms/tools For Sgrna Target Finding and Evaluation Anmentioning

confidence: 99%

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CRISPR/Cas Systems in Genome Editing: Methodologies and Tools for sgRNA Design, Off‐Target Evaluation, and Strategies to Mitigate Off‐Target Effects

et al. 2020

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“…However, the cellular context also plays a role in Cas effector binding and cleavage. SpCas9 can bind to targets containing several mismatches depending on DNA breathing and supercoiling, both in vitro and in vivo (Newton et al 2019;Wu et al 2014). Cas12a can stably bind to targets with mismatches in vitro (Singh et al 2018), but in vivo studies suggest low or no off-target binding ).…”

Section: Discussionmentioning

confidence: 99%

Pervasive off-target and double-stranded DNA nicking by CRISPR-Cas12a

Murugan

Seetharam

Severin

et al. 2019

Preprint

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Cas12a (formerly Cpf1) is an RNA-guided endonuclease in the CRISPR-Cas immune system that can be easily programmed for genome editing. Cas12a can bind and cut dsDNA targets with high specificity in vivo, making it an ideal candidate for precise genome editing applications.This specificity is contradictory to the natural role of Cas12a as an immune effector against rapidly evolving phages. However, the native cleavage specificity and activity remains to be fully understood. We employed high-throughput in vitro cleavage assays to determine and compare the native specificities of three Cas12a orthologs. Surprisingly, we observed pervasive nicking of randomized target libraries, with strong nicking activity observed against targets with up to four mismatches. Nicking and cleavage activities are dependent on mismatch type and position, and vary depending on the Cas12a ortholog and crRNA sequence. Our high-throughput and biochemical analysis further reveal that Cas12a has robust activated non-specific nicking and weak non-specific dsDNA degradation activity in trans. Together, our findings reveal Cas12a

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Cas9 Cuts and Consequences; Detecting, Predicting, and Mitigating CRISPR/Cas9 On‐ and Off‐Target Damage

2020

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Large deletions and genomic re‐arrangements are increasingly recognized as common products of double‐strand break repair at Clustered Regularly Interspaced, Short Palindromic Repeats ‐ CRISPR associated protein 9 (CRISPR/Cas9) on‐target sites. Together with well‐known off‐target editing products from Cas9 target misrecognition, these are important limitations, that need to be addressed. Rigorous assessment of Cas9‐editing is necessary to ensure validity of observed phenotypes in Cas9‐edited cell‐lines and model organisms. Here the mechanisms of Cas9 specificity, and strategies to assess and mitigate unwanted effects of Cas9 editing are reviewed; covering guide‐RNA design, RNA modifications, Cas9 modifications, control of Cas9 activity; computational prediction for off‐targets, and experimental methods for detecting Cas9 cleavage. Although recognition of the prevalence of on‐ and off‐target effects of Cas9 editing has increased in recent years, broader uptake across the gene editing community will be important in determining the specificity of Cas9 across diverse applications and organisms.

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DNA stretching induces Cas9 off-target activity

Cited by 127 publications

References 59 publications

CRISPR/Cas Systems in Genome Editing: Methodologies and Tools for sgRNA Design, Off‐Target Evaluation, and Strategies to Mitigate Off‐Target Effects

CRISPR/Cas Systems in Genome Editing: Methodologies and Tools for sgRNA Design, Off‐Target Evaluation, and Strategies to Mitigate Off‐Target Effects

Pervasive off-target and double-stranded DNA nicking by CRISPR-Cas12a

Cas9 Cuts and Consequences; Detecting, Predicting, and Mitigating CRISPR/Cas9 On‐ and Off‐Target Damage

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