CasKAS: direct profiling of genome-wide dCas9 and Cas9 specificity using ssDNA mapping

Georgi K Marinov; Samuel H Kim; S Tansu Bagdatli; Soon Il Higashino; Alexandro E Trevino; Josh Tycko; Tong Wu; Lacramioara Bintu; Michael C Bassik; Chuan He; Anshul Kundaje; William J Greenleaf

doi:10.1186/s13059-023-02930-z

CasKAS: direct profiling of genome-wide dCas9 and Cas9 specificity using ssDNA mapping

Genome Biol. 2023 Apr 21;24(1):85. doi: 10.1186/s13059-023-02930-z.

Authors

Georgi K Marinov^#¹, Samuel H Kim^#², S Tansu Bagdatli³, Soon Il Higashino³, Alexandro E Trevino^{4

5}, Josh Tycko³, Tong Wu⁶, Lacramioara Bintu⁵, Michael C Bassik^{3

7}, Chuan He^{6

8

9}, Anshul Kundaje^{3

10}, William J Greenleaf^{11

12

13

14}

Affiliations

¹ Department of Genetics, School of Medicine, Stanford University, Stanford, CA, 94305, USA. GKM359@gmail.com.
² Cancer Biology Program, School of Medicine, Stanford University, Stanford, CA, 94305, USA.
³ Department of Genetics, School of Medicine, Stanford University, Stanford, CA, 94305, USA.
⁴ Center for Personal Dynamic Regulomes, Stanford University, Stanford, CA, 94305, USA.
⁵ Department of Bioengineering, Stanford University, Stanford, CA, 94305, USA.
⁶ Department of Chemistry, The University of Chicago, Chicago, IL, 60637, USA.
⁷ Chemistry, Engineering, and Medicine for Human Health (ChEM-H), Stanford University, Stanford, CA, 94305, USA.
⁸ Department of Biochemistry and Molecular Biology and Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL, 60637, USA.
⁹ Howard Hughes Medical Institute, The University of Chicago, Chicago, IL, 60637, USA.
¹⁰ Department of Computer Science, Stanford University, Stanford, CA, 94305, USA.
¹¹ Department of Genetics, School of Medicine, Stanford University, Stanford, CA, 94305, USA. wjg@stanford.edu.
¹² Department of Applied Physics, Stanford University, Stanford, CA, 94305, USA. wjg@stanford.edu.
¹³ Center for Personal Dynamic Regulomes, Stanford University, Stanford, CA, 94305, USA. wjg@stanford.edu.
¹⁴ Chan Zuckerberg Biohub, San Francisco, CA, USA. wjg@stanford.edu.

^# Contributed equally.

Abstract

Detecting and mitigating off-target activity is critical to the practical application of CRISPR-mediated genome and epigenome editing. While numerous methods have been developed to map Cas9 binding specificity genome-wide, they are generally time-consuming and/or expensive, and not applicable to catalytically dead CRISPR enzymes. We have developed CasKAS, a rapid, inexpensive, and facile assay for identifying off-target CRISPR enzyme binding and cleavage by chemically mapping the unwound single-stranded DNA structures formed upon binding of a sgRNA-loaded Cas9 protein. We demonstrate this method in both in vitro and in vivo contexts.

Publication types

Research Support, Non-U.S. Gov't
Research Support, N.I.H., Extramural

MeSH terms

CRISPR-Associated Protein 9 / genetics
CRISPR-Cas Systems*
DNA, Single-Stranded* / genetics
Epigenome
Gene Editing / methods
Genome

Substances

DNA, Single-Stranded
CRISPR-Associated Protein 9

Abstract

Publication types

MeSH terms

Substances

Grants and funding