CRISPR-Cas9 cleavage efficiency correlates strongly with target-sgRNA folding stability: from physical mechanism to off-target assessment

Xu, Xiaojun; Duan, Dongsheng; Chen, Shijie

doi:10.1038/s41598-017-00180-1

Cited by 61 publications

(60 citation statements)

References 35 publications

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“…How Cas12a treats DNAs with deletions or insertions relative to the crRNA is unknown, but structural studies suggest that some R-loop bulges may be accommodated within the protein Stella et al, 2018;Yamano et al, 2016). A deletion within the first 17 positions slows DNA cleavage >10-fold, while the final positions have no affect ( Figure 4D).…”

Section: Analysis Of Wtcas9 Cleavage Productsmentioning

confidence: 99%

Massively parallel kinetic profiling of natural and engineered CRISPR nucleases

Jones

Hawkins

Johnson

et al. 2019

Preprint

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Engineered Streptococcus pyogenes (Sp) Cas9s and Acidaminococcus sp. (As) Cas12a (formerly Cpf1) improve cleavage specificity in human cells. However, the fidelity, enzymatic mechanisms, and cleavage products of emerging CRISPR nucleases have not been profiled systematically across partially mispaired off-target DNA sequences. Here, we describe NucleaSeqnuclease digestion and deep sequencing-a massively parallel platform that measures cleavage kinetics and captures the timeresolved identities of cleaved products for more than ten thousand DNA targets that include mismatches, insertions, and deletions relative to the guide RNA. The binding specificity of each enzyme is measured on the same DNA library via the chip-hybridized association mapping platform (CHAMP). Using this integrated cleavage and binding platform, we profile four SpCas9 variants and AsCas12a. Engineered Cas9s retain wtCas9-like off-target binding but increase cleavage specificity; Cas9-HF1 shows the most dramatic increase in cleavage specificity. Surprisingly, wtCas12a-reported as a more specific nuclease in cells-has cleavage specificity similar to wtCas9 in vitro. Initial cleavage position and subsequent end-trimming vary across nucleases, guide RNA sequences, and position and base identity of mispairs in target DNAs. Using these large datasets, we develop a biophysical model that reveals mechanistic insights into off-target cleavage activities by these nucleases. More broadly, NucleaSeq enables rapid, quantitative, and systematic comparison of the specificities and cleavage products of engineered and natural nucleases.

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Section: Analysis Of Wtcas9 Cleavage Productsmentioning

confidence: 99%

Massively parallel kinetic profiling of natural and engineered CRISPR nucleases

Jones

Hawkins

Johnson

et al. 2019

Preprint

View full text Add to dashboard Cite

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“…Whereas the vast majority of approaches to establish selection rules for target motifs have been generated using association principles, rather than causal relationships, some of these rules have been validated at least using hypothesis‐driven approaches, for example on the topological interaction of gRNA and target DNA (Xu et al ).…”

Section: Selection Of Target Motifsmentioning

confidence: 99%

The CRISPR/Cas revolution continues: From efficient gene editing for crop breeding to plant synthetic biology

Kumlehn

Pietralla

Hensel

et al. 2018

JIPB

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Since the discovery that nucleases of the bacterial CRISPR (clustered regularly interspaced palindromic repeat)‐associated (Cas) system can be used as easily programmable tools for genome engineering, their application massively transformed different areas of plant biology. In this review, we assess the current state of their use for crop breeding to incorporate attractive new agronomical traits into specific cultivars of various crop plants. This can be achieved by the use of Cas9/12 nucleases for double‐strand break induction, resulting in mutations by non‐homologous recombination. Strategies for performing such experiments − from the design of guide RNA to the use of different transformation technologies − are evaluated. Furthermore, we sum up recent developments regarding the use of nuclease‐deficient Cas9/12 proteins, as DNA‐binding moieties for targeting different kinds of enzyme activities to specific sites within the genome. Progress in base deamination, transcriptional induction and transcriptional repression, as well as in imaging in plants, is also discussed. As different Cas9/12 enzymes are at hand, the simultaneous application of various enzyme activities, to multiple genomic sites, is now in reach to redirect plant metabolism in a multifunctional manner and pave the way for a new level of plant synthetic biology.

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“…R-loop formation drives the repositioning and reorientation of the HNH domain into a cleavage-competent state as shown by a crystal structure of Cas9 with a partial R-loop and a cryo-EM reconstruction of a bona-fide Rloop structure (Jiang et al, 2016). Additionally, recent studies have suggested a strong correlation between R-loop stability and CRISPR-Cas9 cleavage efficiency (Xu et al, 2017).…”

Section: Introductionmentioning

confidence: 97%

DNA unwinding is the primary determinant of CRISPR-Cas9 activity

Gong

Johnson

et al. 2017

Preprint

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SummaryBacterial adaptive immunity utilizes RNA-guided surveillance complexes composed of CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) proteins together with CRISPR RNAs (crRNAs) to target foreign nucleic acids for destruction. Cas9, a type II CRISPR-Cas effector complex, can be programed with a single guide RNA that base-pairs with the target strand of dsDNA, displacing the non-target strand to create an R-loop, where the HNH and RuvC nuclease domains can cleave opposing strands. Cas9 has been repurposed for a variety of important genome engineering applications. While many structural and biochemical studies have shed light on the mechanism of Cas9 cleavage, a clear unifying model has yet to emerge.Our detailed kinetic characterization of the enzyme reveals that DNA binding is reversible, Rloop formation is rate-limiting, occurring in two steps, one for each of the nuclease domains.Although the HNH nuclease activity is stimulated by Mg 2+ with a single measureable K d , the RuvC activity requires two distinct Mg 2+ binding events. The specificity constant for cleavage is determined through an induced-fit mechanism as the product of the equilibrium binding affinity for DNA and the rate of R-loop formation.

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CRISPR-Cas9 cleavage efficiency correlates strongly with target-sgRNA folding stability: from physical mechanism to off-target assessment

Cited by 61 publications

References 35 publications

Massively parallel kinetic profiling of natural and engineered CRISPR nucleases

Massively parallel kinetic profiling of natural and engineered CRISPR nucleases

The CRISPR/Cas revolution continues: From efficient gene editing for crop breeding to plant synthetic biology

DNA unwinding is the primary determinant of CRISPR-Cas9 activity

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