Josh Tycko


Systematic discovery of protein functions in human cells to understand gene regulation and enable gene therapy


Contact

Josh Tycko


Contact description



Department of Neurobiology


Harvard Medical School




Josh Tycko


Systematic discovery of protein functions in human cells to understand gene regulation and enable gene therapy



Department of Neurobiology


Harvard Medical School



Identification of Guide-Intrinsic Determinants of Cas9 Specificity


Journal article


Nicholas C. Huston, Josh Tycko, Eric L Tillotson, Christopher J. Wilson, V. Myer, H. Jayaram, Barrett E Steinberg
The CRISPR Journal, 2019

Semantic Scholar DOI PubMedCentral PubMed
Cite

Cite

APA
Huston, N. C., Tycko, J., Tillotson, E. L., Wilson, C. J., Myer, V., Jayaram, H., & Steinberg, B. E. (2019). Identification of Guide-Intrinsic Determinants of Cas9 Specificity. The CRISPR Journal.

Chicago/Turabian
Huston, Nicholas C., Josh Tycko, Eric L Tillotson, Christopher J. Wilson, V. Myer, H. Jayaram, and Barrett E Steinberg. “Identification of Guide-Intrinsic Determinants of Cas9 Specificity.” The CRISPR Journal (2019).

MLA
Huston, Nicholas C., et al. “Identification of Guide-Intrinsic Determinants of Cas9 Specificity.” The CRISPR Journal, 2019.


Abstract

Abstract 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.


Share