O-223 Sperm Genome Editing by CRISPR-Cas9 through an Ooplasm-Mediated Approach

Abstract

Abstract Study question Can we edit the sperm genome by CRISPR-Cas9 through an ooplasmic-mediated approach? Summary answer The ooplasm-mediated technique induced the decondensation of the male gamete nucleus and allowed CRISPR-Cas9 to access the sperm DNA to carry out successful edits. What is known already Previous studies on genome editing using CRISPR-Cas9 have been performed at the S-phase or zygote stage, but challenges were represented by mosaicism and possible off-target edits. To address these issues, gene editing at the gamete level would be ideal. While this may be feasible in oocytes, it appears ambitious in the spermatozoon due to the DNA hypercoiling around the protamine core and the chromatin compaction. In preliminary experiments, permeabilization of sperm membrane allowed penetration of CRISPR-Cas9 to enter the cell, however, genomic editing was proved unsuccessful. Study design, size, duration In the past 5 months, a total of 128 oocytes were divided into 2 groups. To edit exclusively the male genome through oocyte-mediated sperm decondensation (OMSD), a single spermatozoon was injected into an enucleated oocyte to produce a haploid androgenetic embryo. The control cohort consisted of embryos generated through standard ICSI with the established heritable genome editing (HGE) approach. Both groups were treated with CRISPR-Cas9 aiming to knockout Tyr gene to create an albino phenotype. Participants/materials, setting, methods B6D2F1 mice were used to retrieve oocytes and spermatozoa. A cohort of oocytes used for OSMD approach were enucleated. While the intact oocytes were used for HGE control. All oocytes were injected with a single spermatozoon together with CRISPR-Cas9 solution, containing Tyr gRNA. All embryos were cultured up to the 8-cell stage. Individual blastomeres were isolated and sequenced to proof editing. DNA was extracted and amplified for T7E1 analysis to validate genome editing efficiency. Main results and the role of chance Of the 128 oocytes used for the study, 51 were enucleated for the OMSD experiments, while 77 were used for HGE control. After undergoing ICSI with CRISPR-Cas9 solution, 84.3% (43/51) of the OMSD cohort displayed a single male pronucleus and 79.2% (61/77) control HGE fertilized. In a time-lapse microscopy, 95.3% (41/43) of the OMSD experimental group developed to the 2-cell stage, comparable to the HGE control development at 96.7% (59/61). After 48 hours in culture, the HGE control reached 8-cell development at 88.5% (54/61), while the OMSD cohort cleaved at the lower rate at 60.4% (26/43, P<0.001). A 423-bp region around the CRISPR target site was amplified on DNA extracted by isolated blastomere. Gene modification at the target site was confirmed in 33.7% (70/208) of OMSD cohort and 29.7% (88/296) of the HGE control embryos. Limitations, reasons for caution While the experimental observations are still limited, it is important to note that these results may be underestimated since embryos with completely uniform modifications may go undetected by T7E1 assay. The technique needs further refinement to optimize targeting efficiency and support embryo development. Wider implications of the findings Once the genetic editing method is optimized, individual pseudo-blastomeres generated through this OMSD approach can be utilized as male gametes to fertilize oocyte. The generation of live offspring with the corrected haplotype will serve to prove the safety of genome editing through this technique. Trial registration number N/A