Generating CRISPR/Cas9-Derived Mutant Mice by Zygote Cytoplasmic Injection Using an Automatic Microinjector

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) assisted generation of mutant animals has become the method of choice for the elucidation of gene function in development and disease due to the shortened timelines for generation of a desired mutant, the ease of producing materials in comparison to other methodologies (such as embryonic stem cells, ESCs) and the ability to simultaneously target multiple genes in one injection session. Here we describe a step by step protocol, from preparation of materials through to injection and validation of a cytoplasmic injection, which can be used to generate CRISPR mutants. This can be accomplished from start of injection to completion within 2-4 h with high survival and developmental rates of injected zygotes and offers significant advantages over pronuclear and other previously described methodologies for microinjection.

No unexpected CRISPR-Cas9 off-target activity revealed by trio sequencing of gene-edited mice

“…The CRISPR-treated group was split to include five embryos treated with Tyr2F and five embryos treated with Tyr2R, while three untreated embryos from each of three control groups (“Cas9 only”, “No injection” and “Sham injection”) were also collected. Microinjections were performed into the cytoplasm of 1-cell zygotes[ 5 ], which were then briefly cultured to assess viability and then transferred into 0.5 day post coital (d.p.c) pseudopregnant females. Embryos in the “Sham injection” group were microinjected with water only, and the “Cas9 only” embryos were microinjected with Cas9 protein solution only.…”

No unexpected CRISPR-Cas9 off-target activity revealed by trio sequencing of gene-edited mice

“…1a). Microinjections were performed into the cytoplasm of 1-cell zygotes [5], which were then briefly cultured to assess viability and then transferred into 0.5 day post coital (d.p.c) pseudopregnant females. Embryos in the "Sham injection" group were microinjected with water only, and the "Cas9 only" embryos were microinjected with Cas9 protein only.…”

Linkage-specific deubiquitylation by OTUD5 defines an embryonic pathway intolerant to genomic variation

“…Transgenic mice (C57BL/6J) were generated using CRISPR-Cas9 injection and electroporation after isolation of early embryos ( 74 ). For generating Otud5 knock-in mutations, we used a guide RNA (gRNA) p.Gly494Ser 5′-CACCCTGTGCACCAGGTCAG-3′, and for p.Leu352Pro, we used gRNA 5′-CCCCTGGCTTAAATGACGGT-3′ with repair templates including the specific missense mutation.…”