Enhancement of Precise Gene Editing by the Association of Cas9 With Homologous Recombination Factors

Abstract

The CRISPR-Cas9 system is used for genome editing in mammalian cells by introducing double-strand breaks (DSBs) which are predominantly repaired via non-homologous end joining (NHEJ) or to lesser extent by homology-directed repair (HDR). To enhance HDR for improving the introduction of precise genetic modifications, we tested fusion proteins of Cas9 nuclease with HDR effectors to enforce their localization at DSBs. Using a traffic-light DSB repair reporter (TLR) system for the quantitative detection of HDR and NHEJ events in human HEK cells we found that Cas9 fusions with CtIP, Rad52, and Mre11, but not Rad51C promote HDR up to twofold in human cells and significantly reduce NHEJ events. We further compared, as an alternative to the direct fusion with Cas9, two components configurations that associate CtIP fusion proteins with a Cas9-SunTag fusion or with guide RNA that includes MS2 binding loops. We found that the Cas9-CtIP fusion and the MS2-CtIP system, but not the SunTag approach increase the ratio of HDR/NHEJ 4.5-6-fold. Optimal results are obtained by the combined use of Cas9-CtIP and MS2-CtIP, shifting the HDR/NHEJ ratio by a factor of 14.9. Thus, our findings provide a simple and effective tool to promote precise gene modifications in mammalian cells.

Keywords: CRISPR; Cas9; CtIP; gene editing; homologous recombination.

FIGURE 1

Modifying DSB repair pathway choice by association of Cas9 with CtIP. (A) Cas9 induced double-strand breaks are either repaired by HDR or the prevailing NHEJ pathway. DSB repair pathway choice is largely determined by the competition between 53BP1 and BRCA1, triggering either end protection or the CtIP induced resection of DSB ends, followed by the engagement of the NHEJ or HDR pathway. (B) Use of a Cas9-CtIP fusion protein for shifting DSB repair choice toward HDR. (C) Association of Cas9 and CtIP via sgRNA loops that include MS2 phage aptamer sequences bound by fusion proteins of CtIP and the MS2 coat protein. (D) Association of a Cas9-SunTag fusion protein with multiple copies of a SunLigand-CtIP fusion protein.

FIGURE 2

DSB repair modification by Cas9 fusion proteins. (A) The traffic light reporter (TLR) construct indicates DSB repair by NHEJ or HDR and was integrated into the AAVS1 locus of HEK cells (HEK^TLR). Upon induction of a DSB in the defective Venus coding region, RFP is expressed upon NHEJ repair resulting into deletions that shift translation by 2 bp. Venus expression occurs upon HDR with a repair template vector that includes the intact Venus coding region (pTLR-repair). (B) Vectors for the expression of Cas9 or fusion proteins between the N- or C-terminal end of Cas9 and Rad52, RPA1, CtIP wildtype (wt), or the T847E mutant (mut), Mre11A or Rad51C, driven by the CBh promoter. pA – polyadenylation signal. (C) For DSB repair assays HEK^TLR reporter cells were cotransfected with expression vectors for Cas9 or Cas9 fusion proteins, sgRosa26, Blasticidine and pTLR-repair. After 4 days of Blasticidine selection the samples were analyzed by FACS for the presence of Venus and RFP positive cells. (D) Bar graph representation of Venus and RFP positive cells, indicating HDR or NHEJ repair of the TLR reporter, upon transfection with an expression vector for Cas9, or Cas9 in C-terminal (Cter) or N-terminal (Nter) fusion with Rad52, RPA1, CtIP wildtype (wt), CtIP mutant T847E (mut), MRE11A, or Rad51C. Bars show mean values of three independent samples with standard deviation. These values were used to calculate the ratio of Venus⁺ to RFP⁺ cells and p-values (T-test) to determine the significance in levels of Venus⁺ or RFP⁺ cells between samples 1 and 2–9. n.s., not significant, p > 0.05.

FIGURE 3

Cas9 fusion proteins in 53BP1 knockout HEK^TLR cells. (A) For DSB repair assays in HEK^TLR and HEK^TLR cells harboring 53BP1 knockout alleles (HEK^TLR/Δ53BP1), reporter cells were cotransfected in duplicate with expression vectors for Cas9 or Cas9 fusion proteins, sgRosa26, BFP and pTLR-repair. After 4 days the samples were analyzed by flow cytometry (FACS) for the presence of Venus and RFP positive cells indicating repair of the TLR construct by HDR or NHEJ. (B) Bar graph representation of Venus and RFP positive cells, indicating HDR or NHEJ repair of the reporter in HEK^TLR wildtype (53BP1-WT) cells or (C) HEK^TLR/Δ53BP1 (53BP1-KO) cells, upon transfection with an expression vector for Cas9, or Cas9 in C-terminal fusion with MRE11A, Rad52, CtIP wildtype (wt), or the CtIP mutant T847E (mut). Bars show mean values of four (samples 1–3) or three (samples 4, 5) independent samples with standard deviation. These values were used to calculate the ratio of Venus⁺ to RFP⁺ cells and p-values (T-test) to determine the significance in levels of Venus⁺ or RFP⁺ cells between samples 1 and 2–5. n.s., not significant, p > 0.05.

FIGURE 4

Targeting of the beta-2 microglobulin gene (A) The beta-2 microglobulin locus (B2M) was targeted in HEK cells by HDR mediated insertion of a T2A-mCherry coding region placed upstream of the B2M Stop codon and 3′untranslated region (3′-UTR) using a donor vector with 600 bp homology regions (0.6). (B) Four days after the transfection of HEK cells with expression vectors for Cas9 or Cas9 fusion protein, sgRNA against the B2M target region and the donor vector, the frequency of Cherry positive cells was determined by flow cytometry (FACS). (C) Bar graph representation of Cherry positive cells, indicating HDR at the B2M locus upon transfection with an expression vector for Cas9, or Cas9 in C-terminal fusion with MRE11A, Rad52, CtIP wildtype (WT), or the CtIP mutant T847E (mut). Bars show mean values of four independent samples with standard deviation. These values were used to calculate p-values (T-test) to determine the significance in levels of cherry⁺ cells between samples 1 and 2–5.

FIGURE 5

DSB repair modification by MS2-CtIP and SunL-CtIP fusion proteins in HEK^TLR6 reporter cells. (A) Expression vectors for Cas9 or Cas9-SunTag, sgRosa26(MS2), pTLR-donor and CtIP, MS2-CtIP, MS2^di-CtIP, or SunL-CtIP were cotransfected into HEK^TLR6 cells and the frequency of Venus⁺ cells (green columns) and RFP⁺ cells (red columns), indicating DSB repair by HDR or NHEJ, was determined by flow cytometry (FACS) 72 h after transfection. (B) Transfection results are shown as bar graphs representing mean values with standard deviation of triplicate samples. Plasmids selected (+) for individual transfection samples are indicated in the table below. Mean values were used to calculate the ratio of Venus⁺ to RFP⁺ cells and p-values (T-test) to determine the significance in levels of Venus⁺ or RFP⁺ cells between samples 2 and 3–6 (table bottom) and to compare samples 4 and 5 (top horizontal lines). n.s., not significant, p > 0.05. Two independent replicates of the assay were performed that confirmed the results.

FIGURE 6

Comparison of Cas9-CtIP, MS2^di-CtIP, and SunL-CtIP fusion proteins. (A) Expression vectors for Cas9, Cas9-CtIP, or Cas9-SunTag, sgRosa26(MS2), pTLR-donor, and MS2^di-CtIP or SunL-CtIP were cotransfected into HEK^TLR6 cells and the frequency of Venus⁺ cells (green columns) and RFP⁺ cells (red columns), indicating DSB repair by HDR or NHEJ, was determined by FACS analysis 72 h after transfection. (B) Transfection results are shown as bar graphs representing mean values ± standard deviation of triplicate samples. Plasmids selected (+) for individual transfections are indicated as in the table below. Mean values were used to calculate the ratio of Venus⁺ to RFP⁺ cells and p-values (T-test) to determine the significance in levels of Venus⁺ or RFP⁺ cells between samples 5 and 3 or 4 (top horizontal lines). Two independent replicates of the assay were performed that confirmed the results.