Target-Specific Precision of CRISPR-Mediated Genome Editing

Anob M Chakrabarti; Tristan Henser-Brownhill; Josep Monserrat; Anna R Poetsch; Nicholas M Luscombe; Paola Scaffidi

doi:10.1016/j.molcel.2018.11.031

Target-Specific Precision of CRISPR-Mediated Genome Editing

Mol Cell. 2019 Feb 21;73(4):699-713.e6. doi: 10.1016/j.molcel.2018.11.031. Epub 2018 Dec 13.

Authors

Anob M Chakrabarti¹, Tristan Henser-Brownhill², Josep Monserrat², Anna R Poetsch³, Nicholas M Luscombe⁴, Paola Scaffidi⁵

Affiliations

¹ Bioinformatics and Computational Biology Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK; UCL Genetics Institute, Department of Genetics, Evolution and Environment, University College London, London WC1E 6BT, UK.
² Cancer Epigenetics Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK.
³ Bioinformatics and Computational Biology Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK; UCL Genetics Institute, Department of Genetics, Evolution and Environment, University College London, London WC1E 6BT, UK; Okinawa Institute of Science and Technology Graduate University, Onna-son, Okinawa, Japan. Electronic address: arpoetsch@gmail.com.
⁴ Bioinformatics and Computational Biology Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK; UCL Genetics Institute, Department of Genetics, Evolution and Environment, University College London, London WC1E 6BT, UK; Okinawa Institute of Science and Technology Graduate University, Onna-son, Okinawa, Japan.
⁵ Cancer Epigenetics Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK; UCL Cancer Institute, University College London, London WC1E 6DD, UK. Electronic address: paola.scaffidi@crick.ac.uk.

Abstract

The CRISPR-Cas9 system has successfully been adapted to edit the genome of various organisms. However, our ability to predict the editing outcome at specific sites is limited. Here, we examined indel profiles at over 1,000 genomic sites in human cells and uncovered general principles guiding CRISPR-mediated DNA editing. We find that precision of DNA editing (i.e., recurrence of a specific indel) varies considerably among sites, with some targets showing one highly preferred indel and others displaying numerous infrequent indels. Editing precision correlates with editing efficiency and a preference for single-nucleotide homologous insertions. Precise targets and editing outcome can be predicted based on simple rules that mainly depend on the fourth nucleotide upstream of the protospacer adjacent motif (PAM). Indel profiles are robust, but they can be influenced by chromatin features. Our findings have important implications for clinical applications of CRISPR technology and reveal general patterns of broken end joining that can provide insights into DNA repair mechanisms.

Keywords: CRISPR; Cas9; chromatin; genome editing; indel profiles; large-scale; precise; precision; predictability; sgRNA.

Publication types

Comparative Study
Research Support, Non-U.S. Gov't

MeSH terms

CRISPR-Associated Protein 9 / genetics*
CRISPR-Associated Protein 9 / metabolism
CRISPR-Cas Systems*
Cell Proliferation
Chromatin / genetics
Chromatin / metabolism
Chromatin Assembly and Disassembly
Clustered Regularly Interspaced Short Palindromic Repeats*
DNA / genetics*
DNA / metabolism
Gene Deletion*
Gene Editing / methods*
HEK293 Cells
Hep G2 Cells
High-Throughput Nucleotide Sequencing
Humans
Mutagenesis, Insertional*
Nucleotide Motifs
RNA, Guide, CRISPR-Cas Systems / genetics
RNA, Guide, CRISPR-Cas Systems / metabolism

Substances

Chromatin
RNA, Guide, CRISPR-Cas Systems
DNA
CRISPR-Associated Protein 9

Abstract

Publication types

MeSH terms

Substances

Grants and funding