Pairwise library screen systematically interrogates Staphylococcus aureus Cas9 specificity in human cells

Tycko, Josh; Barrera, Luis; Huston, Nicholas C.; Friedland, Ari E.; Wu, Xuebing; Gootenberg, Jonathan S.; Abudayyeh, Omar O.; Myer, Vic E.; Wilson, Christopher J.; Hsu, Patrick

doi:10.1038/s41467-018-05391-2

Cited by 35 publications

(16 citation statements)

References 28 publications

(34 reference statements)

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“…S1H; Kleinstiver et al 2015b). As 20-bp guides are markedly less tolerant of mismatches than longer ones for SaCas9 (type II-A SaCas9 and St1Cas9 share 37% identity), we favor the use of 20-bp guides (Tycko et al 2018). Hence, the flexibility of PAM recognition by St1Cas9 LMD9 enhances its targeting capabilities.…”

Section: Functional Pam Sequences For St1cas9 Lmd9 In Mammalian Cellsmentioning

confidence: 95%

Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9

et al. 2020

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Targeting definite genomic locations using CRISPR-Cas systems requires a set of enzymes with unique protospacer adjacent motif (PAM) compatibilities. To expand this repertoire, we engineered nucleases, cytosine base editors, and adenine base editors from the archetypal Streptococcus thermophilus CRISPR1-Cas9 (St1Cas9) system. We found that St1Cas9 strain variants enable targeting to five distinct A-rich PAMs and provide a structural basis for their specificities. The small size of this ortholog enables expression of the holoenzyme from a single adeno-associated viral vector for in vivo editing applications. Delivery of St1Cas9 to the neonatal liver efficiently rewired metabolic pathways, leading to phenotypic rescue in a mouse model of hereditary tyrosinemia. These robust enzymes expand and complement current editing platforms available for tailoring mammalian genomes.

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Section: Functional Pam Sequences For St1cas9 Lmd9 In Mammalian Cellsmentioning

confidence: 95%

Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9

et al. 2020

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“…More gRNAs (~1,400) were employed in a study that introduced the target and gRNA into cells simultaneously 13 , but the low probability of a gRNA and its corresponding target meeting in the same cell resulted in an average mutation rate of 0.2%, and insufficient data for a comprehensive analysis. An approach introducing gRNA and target in the same synthetic construct has been used for the Cpf1 nuclease 14 , and the Staphylococcus aureus Cas9 enzyme 15 . Both profiled proteins have a shorter RNA scaffold sequence, enabling a simpler library cloning procedure to assess more gRNAs.…”

Section: Introductionmentioning

confidence: 99%

Predicting the mutations generated by repair of Cas9-induced double-strand breaks

et al. 2018

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The DNA mutation produced by cellular repair of a CRISPR/Cas9-generated double-strand break determines its phenotypic effect. It is known that the mutational outcomes are not random, and depend on DNA sequence at the targeted location. Here we systematically study the influence of flanking DNA sequence on repair outcome by measuring the edits generated by >40,000 guide RNAs in synthetic constructs. We performed the experiments in a range of genetic backgrounds and using alternative CRISPR/Cas9 reagents. In total, we gathered data for >109 mutational outcomes. The majority of reproducible mutations are insertions of a single base, short deletions, or longer microhomology-mediated deletions. Each gRNA has an individual cell-line dependent bias toward particular outcomes. We uncover sequence determinants of the produced mutations, and use these to derive a predictor of Cas9 editing outcomes. Improved understanding of sequence repair will allow better design of gene editing experiments.

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“…A previous model exists for S. aureus , which do not correlate well with our guide-specific models ( R 2 < 0.1), likely because this model is universal to all guides and is not focused on modeling multiple mismatches. 20 Our analysis implies that for complex off-targets, RNP-specific models offer improved prediction of nuclease cleavage activity.…”

Section: Resultsmentioning

confidence: 89%

Identification of Guide-Intrinsic Determinants of Cas9 Specificity

et al. 2019

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Considerable effort has been devoted to developing a comprehensive understanding of CRISPR nuclease specificity. In silico predictions and multiple genome-wide cellular and biochemical approaches have revealed a basic understanding of the Cas9 specificity profile. However, none of these approaches has delivered a model that allows accurate prediction of a CRISPR nuclease's ability to cleave a site based entirely on the sequence of the guide RNA (gRNA) and the target. We describe a library-based biochemical assay that directly reports the cleavage efficiency of a particular Cas9–guide complex by measuring both uncleaved and cleaved target molecules over a wide range of mismatched library members. We applied our assay using libraries of targets to evaluate the specificity of Staphylococcus aureus Cas9 under a variety of experimental conditions. Surprisingly, our data show an unexpectedly high variation in the random gRNA:target DNA mismatch tolerance when cleaving with different gRNAs, indicating guide-intrinsic mismatch permissiveness and challenging the assumption of universal specificity models. We use data generated by our assay to create the first off-target, guide-specific cleavage models. The barcoded libraries of targets approach is rapid, highly modular, and capable of generating protein- and guide-specific models, as well as illuminating the biophysics of Cas9 binding versus cutting. These models may be useful in identifying potential off-targets, and the gRNA-intrinsic nature of mismatch tolerance argues for coupling these specificity models with orthogonal methods for a more complete assessment of gRNA specificity.

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Pairwise library screen systematically interrogates Staphylococcus aureus Cas9 specificity in human cells

Cited by 35 publications

References 28 publications

Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9

Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9

Predicting the mutations generated by repair of Cas9-induced double-strand breaks

Identification of Guide-Intrinsic Determinants of Cas9 Specificity

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