Арчи Хмелевский
Science. It works, bitches.
Here we describe the correction of the heterozygous MYBPC3 mutation in human preimplantation embryos with precise CRISPR–Cas9-based targeting accuracy and high homology-directed repair efficiency by activating an endogenous, germline-specific DNA repair response. Induced double-strand breaks (DSBs) at the mutant paternal allele were predominantly repaired using the homologous wild-type maternal gene instead of a synthetic DNA template. By modulating the cell cycle stage at which the DSB was induced, we were able to avoid mosaicism in cleaving embryos and achieve a high yield of homozygous embryos carrying the wild-type MYBPC3 gene without evidence of off-target mutations. The efficiency, accuracy and safety of the approach presented suggest that it has potential to be used for the correction of heritable mutations in human embryos by complementing preimplantation genetic diagnosis. However, much remains to be considered before clinical applications, including the reproducibility of the technique with other heterozygous mutations.
We sought to investigate human gamete and embryo DNA repair mechanisms activated in response to CRISPR–Cas9-induced DSBs. In an effort to demonstrate the proof-of-principle that heterozygous gene mutations can be corrected in human gametes or early embryos, we focused on the MYBPC3 mutation that has been implicated in HCM. Although homozygous mutations with no PGD alternative would have been most desirable for gene correction, generating homozygous human embryos for research purposes is practically impossible. Homozygous MYBPC3 mutations in adults are extremely rare owing to the severity of the clinical symptoms and early onset of the disease. Therefore, we specifically targeted the heterozygous four-base-pair (bp) deletion in the MYBPC3 gene in human zygotes introduced by heterozygous, carrier sperm while oocytes obtained from healthy donors provided the wild-type allele. By accurate analysis of cleaving embryos at the single-cell level, we show high targeting efficiency and specificity of preselected CRISPR–Cas9 constructs. Moreover, DSBs in the mutant paternal MYBPC3 gene were preferentially repaired using the wild-type oocyte allele as a template, suggesting an alternative, germline-specific DNA repair response. Mechanisms responsible for mosaicism in embryos were also investigated and a proposed solution to minimize their occurrence developed—namely the co-injection of sperm and CRISPR–Cas9 components into metaphase II (MII) oocytes.
An adult male patient with well-documented familial HCM caused by a heterozygous dominant 4-bp GAGT deletion (g.9836_9839 del., NC_000011.10) in exon 16 of MYBPC3, currently managed with an implantable cardioverter defibrillator and antiarrhythmic medications, agreed to donate skin, blood and semen samples. Skin fibroblast cultures were expanded and used to generate heterozygous patient induced pluripotent stem cells (iPSCs) as described previously15. Two single-guide RNA (sgRNA)–Cas916, 17, 18 constructs were designed to target this specific MYBPC3∆GAGT deletion (Extended Data Fig. 1a, b) along with two exogenous single-stranded oligodeoxynucleotide (ssODN) templates encoding homology arms to the targeted region (Extended Data Table 1). To differentiate from the wild-type allele, two synonymous single-nucleotide substitutions were introduced into each ssODN template. In addition, ssODN-2 nucleotide substitutions provided an additional restriction enzyme (BstBI) recognition site (Extended Data Fig. 1a, b).
The efficacy and specificity of each construct were tested by transfecting patient iPSCs. Cells were electroporated together with ssODN, Cas9 and sgRNA expression plasmids and subcloned, and the targeted region for each clone was analysed by sequencing (Extended Data Fig. 1c). Of 61 iPSC clones transfected with CRISPR–Cas9-1, 44 (72.1%) were not targeted, as evidenced by the presence of both intact wild-type and intact mutant alleles. Among targeted clones, 10 of 17 (58.8%) were repaired by NHEJ and contained various indels adjacent to the mutation site (Extended Data Fig. 1d, e and Supplementary Table 1). The remaining seven clones were repaired by HDR using ssODN-1 as judged by the presence of the marker nucleotide substitutions. Thus, the total targeting efficiency for CRISPR–Cas9-1 was 27.9% (17/61). Among the targeted clones, only 41.2% (7/17) were repaired by HDR (Extended Data Fig. 1e). The targeting efficiency with CRISPR–Cas9-2 was 13.1% (23/175) and the HDR was considerably lower at 13% (3/23). Of note, among the three HDR-repaired iPSC clones, two were repaired using the ssODN-2 template while the third clone contained intact wild-type sequences in both alleles (Extended Data Fig. 1d, e and Supplementary Table 1), indicating HDR using the wild-type allele.
The wild-type allele in all iPSC clones analysed remained intact, demonstrating high fidelity of sgRNAs.
We also directly compared CRISPR–Cas9-1 and CRISPR–Cas9-2 in patient iPSCs transfected with preassembled Cas9 ribonucleoproteins (RNPs). Targeted deep sequencing demonstrated that CRISPR–Cas9-1 had higher HDR efficiency (Extended Data Fig. 1f). On-target mutations were not detected in wild-type embryonic stem (ES) cells (H9) carrying both wild-type MYBPC3 alleles, demonstrating high specificity of CRISPR–Cas9-1. On the basis of these outcomes, we selected CRISPR–Cas9-1 (hereafter referred to as CRISPR–Cas9), with higher efficiency of HDR-based gene correction, for subsequent studies.