Detailed Phenotypic and Molecular Analyses of Genetically Modified Mice Generated by CRISPR-Cas9-Mediated Editing

Abstract

The bacterial CRISPR-Cas9 system has been adapted for use as a genome editing tool. While several recent reports have indicated that successful genome editing of mice can be achieved, detailed phenotypic and molecular analyses of the mutant animals are limited. Following pronuclear micro-injection of fertilized eggs with either wild-type Cas9 or the nickase mutant (D10A) and single or paired guide RNA (sgRNA) for targeting of the tyrosinase (Tyr) gene, we assessed genome editing in mice using rapid phenotypic readouts (eye and coat color). Mutant mice with insertions or deletions (indels) in Tyr were efficiently generated without detectable off-target cleavage events. Gene correction of a single nucleotide by homologous recombination (HR) could only occur when the sgRNA recognition sites in the donor DNA were modified. Gene repair did not occur if the donor DNA was not modified because Cas9 catalytic activity was completely inhibited. Our results indicate that allelic mosaicism can occur following -Cas9-mediated editing in mice and appears to correlate with sgRNA cleavage efficiency at the single-cell stage. We also show that larger than expected deletions may be overlooked based on the screening strategy employed. An unbiased analysis of all the deleted nucleotides in our experiments revealed that the highest frequencies of nucleotide deletions were clustered around the predicted Cas9 cleavage sites, with slightly broader distributions than expected. Finally, additional analysis of founder mice and their offspring indicate that their general health, fertility, and the transmission of genetic changes were not compromised. These results provide the foundation to interpret and predict the diverse outcomes following CRISPR-Cas9-mediated genome editing experiments in mice.

LysMD3 is a type II membrane protein without an role in the response to a range of pathogens

“…LysMD3 gene trap mutant mice were genotyped using primers listed in Table 1, with GT mutant alleles identified by primers A+C and WT alleles identified by primers B+C. To generate CRISPR/Cas9 modified mLysMD3-deficient mice, B6 embryos were microinjected with mLysMD3 EN gRNA 1 and 2 (Table 1) and Cas9 mRNA and transferred to pseudopregnant recipient female mice, as previously described (54). Mice were screened for loss of exon 2 by Southern blot and targeted alleles verified by Sanger sequencing.…”

A CRISPR Path to Engineering New Genetic Mouse Models for Cardiovascular Research

“…Note that this approach will not detect very large deletions. 137 However, indels in the range of 35–100 nucleotides can easily be resolved in a 2% agarose gel (Supplemental Figure VIIa–VIIb). The PCR products may then be excised from the gel, column purified (Qiagen), and cloned directly into a plasmid (TOPO PCR cloning kit) for Sanger sequence analysis.…”

Inter-homologue repair in fertilized human eggs?

“…During gene editing, NHEJ is usually considered to lead to small indels at DSB sites. However, with appropriate experimental design, long deletions and other events can be detected in cultured cells and in both mouse and pig zygotes [5][6][7] . In Ma et al, genotyping involved amplification of a ~534 bp fragment in which the MYBPC3 ΔGAGT mutation is ~200 bp from a primer-binding site.…”