Precise allele-specific genome editing by spatiotemporal control of CRISPR-Cas9 via pronuclear transplantation

Abstract

Gene-targeted animal models that are generated by injecting Cas9 and sgRNAs into zygotes are often accompanied by undesired double-strand break (DSB)-induced byproducts and random biallelic targeting due to uncontrollable Cas9 targeting activity. Here, we establish a parental allele-specific gene-targeting (Past-CRISPR) method, based on the detailed observation that pronuclear transfer-mediated cytoplasmic dilution can effectively terminate Cas9 activity. We apply this method in embryos to efficiently target the given parental alleles of a gene of interest and observed little genomic mosaicism because of the spatiotemporal control of Cas9 activity. This method allows us to rapidly explore the function of individual parent-of-origin effects and to construct animal models with a single genomic change. More importantly, Past-CRISPR could also be used for therapeutic applications or disease model construction.

Establishment, characterization, and validation of novel porcine embryonic fibroblasts as a potential source for genetic modification

“…Extensive reports on the high rates of generating genotypic mosaicism induced in CRISPR/Cas9 mediated gene-edited animals generated by zygote injection have already been published ( Park et al, 2016 ; Zhou et al, 2016 ; Burkard et al, 2017 ; Hennig et al, 2022 ). From a technical perspective, previous reports of generating GE animals by zygotic injections among various species with the goal of reducing mosaicism have met with limited success ( Hashimoto et al, 2016 ; Ma et al, 2017 ; Vilarino et al, 2017 ; Li et al, 2020 ). Despite the high targeting efficiency in the zygotes and relative simplicity of the procedure, a great deal of work remains to be performed on this issue of mosaicism and inheritance of intact alleles, confounding the phenotype of resulting mutations and alleles among the G0 founders.…”

Evaluation of guide-free Cas9-induced genomic damage and transcriptome changes in pig embryos

“… 5 , 6 Previously, off-target mutations have been major safety concerns for the practice of germline genome editing with the CRISPR-Cas9 system, especially for human germline gene therapy, and numerous efforts have been exerted to minimize off-target effects. 7 , 8 , 9 However, beyond off-target issues, recent studies reported that the expression of Cas9 protein alone can lead to negative effects on cells, which raised another serious safety concern regarding the application of the CRISPR-Cas9 system. 10 , 11 , 12 , 13 , 14 In human cells, SpyCas9 can cause DNA damage, most likely because of its single-strand DNA cleavage activity in the presence of metal ions, without sequence preference.…”