Timed inhibition of CDC7 increases CRISPR-Cas9 mediated templated repair

Wienert B, Nguyen DN, Guenther A, Feng SJ, Locke MN, Wyman SK, Shin J, Kazane KR, Gregory GL, Carter MAM, Wright F, Conklin BR, Marson A, Richardson CD, Corn JE
Source: Nat Commun.
Publication Date: (2020)
Issue: 11(1): 2109
Research Area:
Basic Research
Cells used in publication:
Species: human
Tissue Origin: kidney
Species: human
Tissue Origin: cervix
T cell, human stim.
Species: human
Tissue Origin: blood
Fibroblast, dermal(NHDF-Ad), human adult
Species: human
Tissue Origin: dermal
HCT 116
Species: human
Tissue Origin: colon
Species: human
Tissue Origin: brain
Induced Pluripotent Stem Cell (iPS), human
Species: human
Tissue Origin:
Culture Media:
4D-Nucleofector® 96-well Systems

 Streptococcus pyogenes Cas9 (pMJ915, Addgene #69090) with two nuclear localization signal peptides and an HA tag at the C-terminus was expressed in Rosetta2 DE3 (UC Berkeley Marcolab) cells, Cas9-NLS was obtained from the QB3 MacroLab at UC Berkeley. 

sgRNAs were synthesized by Synthego as modified gRNAs with 2'-O-methyl analogs and 3' phosphorothioate internucleotide linkages at the first three 5' and 3' terminal RNA residues using protospacer sequences.

crRNAs/tracrRNAs were chemically synthesized (Edit-R, Dharmacon Horizon) using protospacer sequences. ssDonors were obtained by ordering unmodified Ultramer oligonucleotides (IDT). dsDonor was obtained by purifying plasmid DNA from bacterial cultures containing the indicated plasmid (Qiagen) or by SPRI purification of long double-stranded PCR amplicons.

Cas9 RNP assembly and nucleofection: Fifty pmoles of sgRNA was diluted using Cas9 buffer (20?nM HEPES [pH 7.5]), 150mM KCl, 1mM MgCl2, 10% glycerol, and 1mM TCEP) or water. 1.25µL of 40mM Cas9-2xNLS (50 pmoles) was slowly mixed in, and the resulting mixture was incubated for 5min at room temperature to allow for RNP formation. After incubation, either 0.5µL of 100µM ssDonor or 1.5 or 2µg of plasmid DNA was introduced and mixed by pipetting. The total volume of RNP solution was 5ml, where the volume of Cas9 buffer was adjusted to account for volume differences between ssDonor and plasmid DNA. Between 1e+05 and 2e+05 cells were harvested, washed once in PBS, and resuspended in 15µL of nucleofection buffer (Lonza, Basel, Switzerland). Five microliter of RNP mixture was added to 15µL of cell suspensions. Reaction mixtures were electroporated in Lonza 4D nucleocuvettes, incubated in the nucleocuvette at room temperature for five minutes, and transferred to culture dishes containing pre-warmed media. Large-scale nucleofections were performed by splitting cultures and conducting multiple parallel nucleofections.

Editing outcomes were measured four days post-nucleofection by flow cytometry or by amplicon sequencing. Resuspension buffer and electroporation conditions were the following from each cell line: K562 in buffer SF with FF-120, HEK293 in buffer SF with DS-150, T cells in buffer P3 with EH-115, primary mouse glial cells in buffer P3 with DS-112, HDLECs in buffer P3 with CA-137 HCT116s in buffer SE with EN-113, and HeLa cells in buffer SE with CN-114,U251 cells in buffer SE with DS130, iPSCs in buffer P3 with DS-138, HDFs in buffer P3 with DS-150 and HSPCs in buffer P3 with ER-100.


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.