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.
Highlights
Gene-targeted animal models that are generated by injecting Cas[9] and sgRNAs into zygotes are often accompanied by undesired double-strand break (DSB)-induced byproducts and random biallelic targeting due to uncontrollable Cas[9] targeting activity
The injection of MII oocytes with Cas[9] RNPs (Cas9:sgRNA ribonucleotide protein complexes) at fertilization has been reported to lead to lower mosaicism[20,21], so we examined the same geneediting strategy by targeting Anapc[2]
Cas[9] targeting activity could endure into the two-cell or later stages, which could lead to the additional undirected genomic editing during embryonic development, which may be the reason for the high rates of generating genotypic mosaicism
Summary
Gene-targeted animal models that are generated by injecting Cas[9] and sgRNAs into zygotes are often accompanied by undesired double-strand break (DSB)-induced byproducts and random biallelic targeting due to uncontrollable Cas[9] targeting activity. 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 Cas[9] 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 Cas[9] activity. Parental allele-specific genomic sites cannot be selectively recognized and modified by the traditional CRISPRCas[9] system These defects reveal that a lack of control over Cas[9] activity limits the rapid and economical properties of CRISPR-Cas[9] for research on gene function and for clinical application[12,15,16]. This spatially improves allele selectively to achieve highly efficient parental allele-specific gene editing, and it temporally restricts Cas[9] activity to a narrow window, which can dramatically reduce mosaicism
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