Efficient embryonic homozygous gene conversion via RAD51-enhanced interhomolog repair

Abstract

Searching for factors to improve knockin efficiency for therapeutic applications, biotechnology, and generation of non-human primate models of disease, we found that the strand exchange protein RAD51 can significantly increase Cas9-mediated homozygous knockin in mouse embryos through an interhomolog repair (IHR) mechanism. IHR is a hallmark of meiosis but only occurs at low frequencies in somatic cells, and its occurrence in zygotes is controversial. Using multiple approaches, we provide evidence for an endogenous IHR mechanism in the early embryo that can be enhanced by RAD51. This process can be harnessed to generate homozygotes from wild-type zygotes using exogenous donors and to convert heterozygous alleles into homozygous alleles without exogenous templates. Furthermore, we identify additional IHR-promoting factors and describe features of IHR events. Together, our findings show conclusive evidence for IHR in mouse embryos and describe an efficient method for enhanced gene conversion.

Keywords: BCCIP; CRISPR; DNA repair; RAD51; USP1; WDR48; gene conversion; genome editing; homologous recombination; interhomolog repair.

Figure 1.. Efficient homozygous knock-in with RAD51

(A) Schematic of the targeted locus and HR donor for generating Chd2^RH mutant mice. Donor contains both the c.5051G>A point mutation and a synonymous mutation in the relevant PAM site (HA=homology arm). (B) Example chromatograms of wildtype (top) and Chd2^RH/RH animals (bottom). (C) Overall KI efficiency observed in F₀ pups derived from either cytoplasmic (CPI) or pronuclear (PNI) injection (pups with ≥1 KI allele/total pups). (D) Homozygous KI rates observed in F₀ pups generated by either CPI or PNI, shown as percentage of all pups or pups positive for KI. (E) Genotyping results from F₁ pups derived from crosses between F₀ Chd2^RH/RH animals and wild-type animals.

Figure 2.. RAD51 enhances homozygous KI efficiency at multiple loci

(A) Quantification of KI efficiency (left) and homozygous KI efficiency in embryos generated by Chd2^RH PNI with or without RAD51. All RH/RH includes mosaic embryos and denotes embryos with ≥3:2 ratio of mutant allele to wildtype allele (one-sided Fisher’s exact test). (B) Schematic of knock-in strategy for the albinism-associated Tyr^C89S mutation. (C) Representative examples of Tyr^+/+ (black) and Tyr^C89S/C89S (white) littermates derived from injections using exogenous RAD51 protein. (D) Representative chromatograms from Tyr^+/+ (top) and Tyr^C89S/C89S animals. (E) Genotyping of F₀ animals from injections targeting the Tyr^C89S locus with or without RAD51 (one-sided Fisher’s exact test). *p<0.05, ***p<0.001, n.s. p>0.05

Figure 3.. RAD51 enhances interhomolog repair.

(A) Schematic of strategy for testing evaluating embryonic IHR. Wild-type oocytes were fertilized in vitro by sperm collected from Chd2^RH/RH males and cultured for ~8 hours. PNI was then performed with Cas9 protein, tracrRNA, and crRNA specifically targeting the wild-type maternal allele. Injected embryos were then cultured for 2–3 days and collected at the morula or blastocyst stage. Half of the purified DNA was used for nested PCR and Sanger sequencing and the other half was used for multiplex PCR and subsequent qPCR to analyze genomic copy number at the Chd2 editing locus. (B) Representative chromatograms showing the wild-type reference sequence (Ref., top), an uninjected Chd2^RH/+ embryo generated by IVF (Uninj., middle), and a pure Chd2^RH/RH homozygous mutant (IHR, bottom) generated through IHR. (C) Quantification of Sanger sequencing results from IVF-derived embryos edited with or without RAD51. ‘All IHR’ includes mosaic embryos and was determined by identifying embryos with ≥3:2 ratio of R1684H allele to all other alleles (one-sided Fisher’s exact test). Pure IHR denotes embryos without mosaicism (p=0.018, one-tailed chi-square test). (D) Genomic qPCR targeting the Chd2^RH locus in embryos carrying a large heterozygous deletion (Chd2^del/+) as a control for copy-number sensitivity (lanes 1–2, unpaired t-test, n=3 embryos, error bars=SEM). Lanes 3–13 show genomic qPCR for the Chd2^RH locus using DNA from an uninjected embryo and 10 randomly selected pure homozygous Chd2^RH embryos (p>0.05, unpaired t-test, n=4 technical replicates per sample, error bars=SEM). (E) F₁ genotyping results of litters derived from crosses between wild-type mice and 5 pure Chd2^RH/RH F₀ animals generated by the strategy described in (A), including RAD51. *p<0.05, **p<0.01, ****p<0.0001

Figure 4.. RAD51 Localizes to Cas9-Induced DSBs During G2 in Zygotes.

(A) Representative images of RAD51 immunocytochemistry in G1/S, S/G2, and M-phase zygotes injected with Cas9 and RAD51. Arrowheads indicate RAD51 puncta. Dotted lines highlight pronuclear nucleoli. (Scale bar=10μm) (B) Quantification of zygotes positive for RAD51 puncta (one-sided Fisher’s exact test). ***p<0.001, ****p<0.0001, n.s. p>0.05

Figure 5.. RAD51 Enhances Interhomolog Repair at Loci on Multiple Chromosomes.

(A-C) Genotyping of embryos at the Mecp, Acs16, and Kcnb2 loci. Representative chromatograms of unedited and pure IHR embryos shown below graphs. Red letters denote SNVs and underlined bases represent deletions. All IHR denotes embryos with ≥3:2 ratio of the IHR allele to all other alleles. Pure IHR denotes non-mosaic IHR (one-sided Fisher’s exact test). *p<0.05, **p<0.01

Figure 6.. RAD51 G151D and BCCIP promote zygotic IHR and suppress indel formation.

(A) Quantification of IHR efficiency in blastocysts derived Chd2^RH/+ zygotes injected with Cas9/crRNA/tracrRNA and the indicated proteins. Quantification includes mosaic embryos (one-sided Fisher’s exact test). (B) Quantification of IHR efficiency in blastocysts derived Chd2^RH/+ zygotes injected with Cas9/crRNA/tracrRNA and the indicated RAD51 variants (one-sided Fisher’s exact test). (C) Quantification of editing efficiency at the Chd2^RH locus in embryos injected with Cas9/crRNA/tracrRNA and the indicated RAD51 variants (one-sided Fisher’s exact test). (D) In vitro Cas9 nuclease activity assay using a PCR amplimer of the Chd2^RH locus and Cas9 alone or co-incubated with either RAD51 G151D or BCCIP. (E) Quantification of the percent of total injected embryos carrying an indel after injection with the specified proteins (one-sided Fisher’s exact test). (F) Quantification of the ratio of indel-positive embryos to IHR-positive embryos derived from injections with the specified proteins (one-sided Fisher’s exact test). *p<0.05, **p<0.01, ****p<0.0001

Figure 7.. IHR Induces Conversion of Long Recombination Tracts.

(A) Schematic of genotyping strategies for Acs16 and Kcnb2 loci. Black arrows denote locations of primers for local PCR of SNPs tiled across regions. Red arrows denote locations of primers for long-range PCR and TOPO cloning. Vertical lines denote approximate locations of SNPs. (B) Summary of genotyping results from local PCR of tiled SNPs. Pink boxes denote homozygous A/J alleles and mixed-color boxes denote heterozygous alleles with A/J and C57BL/6J SNPs. (C) Summary of genotyping results from TOPO cloning of long-range PCR products. Mixed-color boxes are used for any sites with at least one clone of the indicated allele. Asterisks denote embryos with low level of an allele, indicating mosaicism (see also Figure S5).