Day 1–3: ~4 week-old females are superovulated, first by PMSG injection, 46–48 hours later by hCG injection, before being housed with stud males for breeding. (b) A graphic overview of CRISPR-EZ workflow. Design sgRNAs, HDR donor oligos, and editing validation assays prior to CRISPR-EZ experiments. Multiple sgRNAs can be used to engineer a genomic deletion by NHEJ repair. A single sgRNA can be used to create a small indel via NHEJ repair or in conjunction with a ssODNs to create a precision mutation or a small insertion by HDR. (a) An illustration of the most successful CRISPR-EZ editing strategies. Altogether, CRISPR-EZ is a simple, economic, efficient, and high-throughput technology that is potentially applicable to other mammalian species.
Using CRISPR-EZ, a graduate-level researcher with basic embryo-manipulation skills can obtain genetically modified mice in 6 weeks. Here, we provide an optimized protocol covering sgRNA synthesis, embryo collection, RNP electroporation, mouse generation, and genotyping strategies. In a side-by-side comparison in the high-throughput KnockOut Mouse Project (KOMP) pipeline, CRISPR-EZ consistently outperformed microinjection. In C57BL/6J and C57BL/6N mouse strains, CRISPR-EZ achieves 100% delivery of Cas9/single-guide RNA (sgRNA) RNPs, facilitating indel mutations (insertions or deletions), exon deletions, point mutations, and small insertions. We thus developed CRISPR ribonucleoprotein (RNP) electroporation of zygotes (CRISPR-EZ), an electroporation-based technology that outperforms pronuclear and cytoplasmic microinjection in efficiency, simplicity, cost, and throughput. CRISPR/Cas9 technology has transformed mouse genome editing with unprecedented precision, efficiency, and ease however, the current practice of microinjecting CRISPR reagents into pronuclear-stage embryos remains rate-limiting.
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