O-299 Ooplasm-mediated sperm nuclear decondensation for genome editing of the mammalian male gamete

Study question Can CRISPR-Cas9 efficiently edit a specific gene in a single spermatozoon using an ooplasm-mediated mechanism? Summary answer The ooplasm-mediated approach induced the decondensation of the male gamete nucleus enabling CRISPR-Cas9 access to the sperm genome successfully editing the coat pigment gene. What is known already CRISPR-Cas9 has been applied to edit somatic and germline cells through microinjection, electroporation, or transfection. Early heritable genome editing (HGE) efforts focused on S-phase or zygote stage but encountered mosaicism and loss of heterozygosity. To overcome these limitations, editing at the gamete level emerged as a promising strategy. While genome editing in oocytes is considered feasible, sperm DNA editing presents greater difficulty due to highly compacted chromatin structure surrounding the protamine cores. Attempts in sperm membrane permeabilization facilitated CRISPR-Cas9 entry, but genomic editing was unsuccessful. We aim to overcome chromatin condensation by using intrinsic ooplasmic machinery to decondense sperm genome. Study design, size, duration Over the past year, oocytes were divided into two groups: one for embryo genome editing and the other for sperm genome editing via Oocyte-Mediated Sperm Decondensation (OMSD). In the OMSD approach, a single spermatozoon was injected into an enucleated oocyte to generate haploid embryo with only paternal DNA. CRISPR-Cas9 targeting the Tyr gene with Tyr-specific sgRNA was utilized in both cohorts to induce a knockout, resulting in an albino phenotype. Participants/materials, setting, methods B6D2F1 mice were used to retrieve metaphase II oocytes and spermatozoa. A subset of oocytes was enucleated for the OMSD approach, while other oocytes remained intact. All oocytes were injected with a spermatozoon, together with Tyr-specific sgRNA and Cas9 protein. Embryos were cultured to 8-cell stage, and genomic DNA was analyzed by T7E1 cleavage assay to assess genome editing efficiency. Control embryos were generated from intact oocytes using the standard HGE approach. Main results and the role of chance In this study, a total of 173 oocytes were used, with 46 intact oocytes undergoing standard embryo genome editing and 127 enucleated oocytes for OMSD experiments. After Piezo-ICSI with a CRISPR-Cas9 solution, fertilization of intact oocytes occurred at a rate of 82.6% (38/46). Following 48 hours of culture, 86.8% (33/38) of diploid embryos reached the 8-cell stage. Amplification of the 584-bp region of the target site from extracted DNA confirmed gene modification in 90.9% (30/33) of the edited diploid embryos. In the OMSD cohort, enucleated oocytes fertilized as 1PN derived pseudo-zygote at a rate of 54.2% (58/107), and cleavage was lower, with only 34.5% (20/58) progressing to the 8-cell stage. Sperm genome modification was confirmed in 71.0% (22/31) of the embryos. While enucleation reduces cleavage, the overall gene modification efficiency remains high. Transfer of edited blastocysts resulted in an offspring correcting the coat pigmentation. Limitations, reasons for caution Limitations include small sample size and potential variability of CRISPR-Cas9 editing, which may lead to underestimation of results, as uniform modifications may go undetected by T7E1 assay. Further technique refinement is necessary to optimize targeting efficiency and embryo development. Additionally, epigenetic modifications and offspring health have yet to be evaluated. Wider implications of the findings Sperm genome decondensation through OMSD enabled CRISPR-Cas9 to actually edit the male gamete. Moreover, this method offers a reliable mean to uniformly target and edit deleterious gene that may be present in a male individual. Trial registration number No