Altered DNA repair pathway engagement by engineered CRISPR-Cas9 nucleases

Abstract

CRISPR-Cas9 introduces targeted DNA breaks that engage competing DNA repair pathways, producing a spectrum of imprecise insertion/deletion mutations (indels) and precise templated mutations (precise edits). The relative frequencies of these pathways are thought to primarily depend on genomic sequence and cell state contexts, limiting control over mutational outcomes. Here, we report that engineered Cas9 nucleases that create different DNA break structures engage competing repair pathways at dramatically altered frequencies. We accordingly designed a Cas9 variant (vCas9) that produces breaks which suppress otherwise dominant nonhomologous end-joining (NHEJ) repair. Instead, breaks created by vCas9 are predominantly repaired by pathways utilizing homologous sequences, specifically microhomology-mediated end-joining (MMEJ) and homology-directed repair (HDR). Consequently, vCas9 enables efficient precise editing through HDR or MMEJ while suppressing indels caused by NHEJ in dividing and nondividing cells. These findings establish a paradigm of targeted nucleases custom-designed for specific mutational applications.

Keywords: CRISPR; DNA repair; genome editing; molecular engineering; precise editing.

Fig. 1.

Engineered Cas9 variants that make staggered cuts have altered repair outcomes. (A) Model of the balance between NHEJ and HDR or MMEJ repair pathways for Cas9 variants. (B) Cas9 residues at the interface with the substrate DNA strands that were selected for alanine substitution and altered repair pathway screening. The mutated residues (red) are located in either the mobile HNH domain (green) or the immobile RuvC domain (blue), which are connected by linker segments (yellow). (C) Design of a guide RNA (gRNA) and HDR template to introduce precise edits. (D) DNA break positions in the target strand (TS) and nontarget strand (NTS) for engineered Cas9 single-mutant variants. (E) Screen of precise editing and indel frequencies for engineered Cas9 single-mutant variants using an HDR template. (F) Correlation between staggered cut frequency and precise editing frequency across Cas9 variants from the alanine substitution screen. *indicates P < 0.05 for precise editing frequency compared to wild-type Cas9. Data were analyzed by deep sequencing and represent means of n = 3 independent replicates with SEs.

Fig. 2.

Design of Cas9 variants with altered repair pathway frequencies. (A) Design of gRNAs and HDR templates to introduce precise edits. (B) Screen of precise editing and indel frequencies for engineered Cas9 double-mutant variants using an HDR template. (C) Cas9 residues proximal to R976 that were selected for arginine substitution and activity rescue screening. The mutated residues (teal) are located near the nontarget substrate DNA strand (pink). (D and E) Screen of precise editing and indel frequencies for engineered Cas9 (D) triple-mutant and (E) quadruple-mutant variants using an HDR template. *indicates P < 0.05 for precise editing frequency compared to wild-type Cas9 in B or for total editing frequency compared to R976A-K1003A in D and E. Data were analyzed by deep sequencing and represent means of n = 3 independent replicates with SEs.

Fig. 3.

Staggered cutting by vCas9 suppresses NHEJ and makes MMEJ or HDR dominant. (A) DNA break positions in the target strand (TS) and nontarget strand (NTS) for engineered Cas9 variants. (B) Distributions of indel sizes induced. (C) Mean indel size induced at several loci. (D and E) Frequencies of repair pathways engaged (D) without (noT) and (E) with (T) a repair template at several loci. (F) Degree of depletion by a repair template for indels of different sizes averaged over several loci. The fraction depleted is increased by vCas9 at all indel sizes. *indicates P < 0.05 for indel size compared to wild-type Cas9 in B and C, for NHEJ frequency compared to wild-type Cas9 in D and E, or for indel depletion compared to wild-type Cas9 in F. Data were analyzed by deep sequencing and represent means of n = 3 independent replicates with SEs.

Fig. 4.

vCas9 enhances precise editing and suppresses indels across diverse editing contexts. (A) Precise editing and indel frequencies using small replacement (<10 bp) HDR templates at several loci. (B) Frequency of precise gene conversion from GFP to BFP. (C) Flow cytometry plots showing BFP-positive (indicating precise edits), nonfluorescent (indicating indels), and GFP-positive (indicating unedited cells) cell population fractions. (D) Design of a gRNA and HDR template to introduce a large insertion. (E) Design of a gRNA to induce untemplated collapse of duplications. (F) Precise editing and indel frequencies using large insertion (~50 bp) templates at several loci. (G) Precise editing and indel frequencies for untemplated collapse of duplications (10 to 20 bp) at several loci. *indicates P < 0.05 for precise editing frequency compared to wild-type Cas9. Data were analyzed by deep sequencing in A, F, and G or flow cytometry in B and C and represent means of n = 3 independent replicates with SEs.

Fig. 5.

vCas9 enables efficient precise editing through MMEJ in dividing and nondividing cells. (A) Design of a gRNA and MDR template to introduce a precise edit. (B) Precise editing and indel frequencies using MDR templates in dividing HEK293T cells. (C) Precise editing and indel frequencies using MDR templates in nondividing (quiescent) primary human dermal fibroblasts. *indicates P < 0.05 for precise editing frequency compared to wild-type Cas9. Data were analyzed by deep sequencing and represent means of n = 3 independent replicates with SEs.

Fig. 6.

Model for how engineered Cas9 variants alter repair outcomes. (A) Cas9 primarily produces blunt cuts, which promote DNA end-joining while limiting resection. This leads to repair dominated by NHEJ over MMEJ and HDR. (B) Cas9 variants like vCas9 create staggered cuts, which promote DNA resection and inhibit end-joining. This enhances repair by MMEJ and HDR while suppressing NHEJ.