Timed inhibition of CDC7 increases CRISPR-Cas9 mediated templated repair.
Beeke WienertDavid N NguyenAlexis GuentherSharon J FengMelissa N LockeStacia K WymanJiyung ShinKatelynn R KazaneGeorgia L GregoryMatthew A M CarterFrancis WrightBruce R ConklinAlexander MarsonChristopher D RichardsonJacob E CornPublished in: Nature communications (2020)
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.
Keyphrases
- crispr cas
- genome editing
- genome wide
- small molecule
- circulating tumor
- copy number
- cell free
- single molecule
- dna methylation
- escherichia coli
- metabolic syndrome
- clinical trial
- cell cycle
- mesenchymal stem cells
- nucleic acid
- single cell
- oxidative stress
- adipose tissue
- skeletal muscle
- circulating tumor cells
- endothelial cells
- cell proliferation
- study protocol
- genome wide analysis
- bioinformatics analysis