Timed inhibition of CDC7 increases CRISPR-Cas9 mediated templated repair

Beeke Wienert; David N Nguyen; Alexis Guenther; Sharon J Feng; Melissa N Locke; Stacia K Wyman; Jiyung Shin; Katelynn R Kazane; Georgia L Gregory; Matthew A M Carter; Francis Wright; Bruce R Conklin; Alex Marson; Chris D Richardson; Jacob E Corn

doi:10.1038/s41467-020-15845-1

Timed inhibition of CDC7 increases CRISPR-Cas9 mediated templated repair

Nat Commun. 2020 Apr 30;11(1):2109. doi: 10.1038/s41467-020-15845-1.

Authors

Beeke Wienert^#^{1

2

3}, David N Nguyen^#^{4

5

6}, Alexis Guenther⁷, Sharon J Feng^{1

2}, Melissa N Locke², Stacia K Wyman¹, Jiyung Shin⁸, Katelynn R Kazane^{1

2}, Georgia L Gregory³, Matthew A M Carter³, Francis Wright⁴, Bruce R Conklin^{3

9}, Alex Marson^{1

4

5

6

10

11}, Chris D Richardson^{12

13

14}, Jacob E Corn^{15

16

17}

Affiliations

¹ Innovative Genomics Institute, University of California, Berkeley, CA, 94703, USA.
² Department of Molecular and Cell Biology, University of California, Berkeley, CA, 94703, USA.
³ Gladstone Institutes, San Francisco, CA, 94158, USA.
⁴ Department of Microbiology and Immunology, University of California, San Francisco, CA, 94143, USA.
⁵ Diabetes Center, University of California, San Francisco, CA, 94143, USA.
⁶ Department of Medicine, University of California, San Francisco, CA, 94143, USA.
⁷ Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, CA, 93106, USA.
⁸ Department of Biology, Institute of Molecular Health Sciences, ETH Zürich, 8093, Zurich, Switzerland.
⁹ Departments of Medicine, Ophthalmology, and Pharmacology, University of California, San Francisco, CA, 94143, USA.
¹⁰ Parker Institute for Cancer Immunotherapy, San Francisco, CA, 94129, USA.
¹¹ Chan Zuckerberg Biohub, San Francisco, CA, 94158, USA.
¹² Innovative Genomics Institute, University of California, Berkeley, CA, 94703, USA. chris.richardson@lifesci.ucsb.edu.
¹³ Department of Molecular and Cell Biology, University of California, Berkeley, CA, 94703, USA. chris.richardson@lifesci.ucsb.edu.
¹⁴ Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, CA, 93106, USA. chris.richardson@lifesci.ucsb.edu.
¹⁵ Innovative Genomics Institute, University of California, Berkeley, CA, 94703, USA. jacob.corn@biol.ethz.ch.
¹⁶ Department of Molecular and Cell Biology, University of California, Berkeley, CA, 94703, USA. jacob.corn@biol.ethz.ch.
¹⁷ Department of Biology, Institute of Molecular Health Sciences, ETH Zürich, 8093, Zurich, Switzerland. jacob.corn@biol.ethz.ch.

^# Contributed equally.

Abstract

Repair of double strand DNA breaks (DSBs) can result in gene disruption or gene modification via homology directed repair (HDR) from donor DNA. Altering cellular responses to DSBs may rebalance editing outcomes towards HDR and away from other repair outcomes. Here, we utilize a pooled CRISPR screen to define host cell involvement in HDR between a Cas9 DSB and a plasmid double stranded donor DNA (dsDonor). We find that the Fanconi Anemia (FA) pathway is required for dsDonor HDR and that other genes act to repress HDR. Small molecule inhibition of one of these repressors, CDC7, by XL413 and other inhibitors increases the efficiency of HDR by up to 3.5 fold in many contexts, including primary T cells. XL413 stimulates HDR during a reversible slowing of S-phase that is unexplored for Cas9-induced HDR. We anticipate that XL413 and other such rationally developed inhibitors will be useful tools for gene modification.

Publication types

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

MeSH terms

CRISPR-Cas Systems*
Cell Cycle Proteins / antagonists & inhibitors*
Cell Cycle Proteins / genetics*
DNA Breaks, Double-Stranded
Gene Editing
Genetic Engineering / methods
HCT116 Cells
HEK293 Cells
HeLa Cells
Homologous Recombination
Humans
K562 Cells
Phenotype
Protein Serine-Threonine Kinases / antagonists & inhibitors*
Protein Serine-Threonine Kinases / genetics*
RNA, Guide, CRISPR-Cas Systems / metabolism
Recombinational DNA Repair*
S Phase

Substances

Cell Cycle Proteins
RNA, Guide, CRISPR-Cas Systems
CDC7 protein, human
Protein Serine-Threonine Kinases

Abstract

Publication types

MeSH terms

Substances

Grants and funding