Impact of Essential Genes on the Success of Genome Editing Experiments Generating 3,313 New Genetically Engineered Mouse Lines

Abstract

The International Mouse Phenotyping Consortium (IMPC) is generating and phenotyping null mutations for every protein-coding gene in the mouse. The IMPC now uses Cas9, a programmable RNA-guided nuclease that has revolutionized mouse genome editing and increased capacity and flexibility to efficiently generate null alleles in the C57BL/6N strain. In addition to being a valuable novel and accessible research resource, the production of >3,300 knockout mouse lines using comparable protocols provides a rich dataset to analyze experimental and biological variables affecting in vivo null allele engineering with Cas9. Mouse line production has two critical steps - generation of founders with the desired allele and germline transmission (GLT) of that allele from founders to offspring. Our analysis identified that whether a gene is essential for viability was the primary factor influencing successful production of null alleles. Collectively, our findings provide best practice recommendations for generating null alleles in mice using Cas9; these recommendations may be applicable to other allele types and species.

Baat Gene Knockout Alters Post-Natal Development, the Gut Microbiome, and Reveals Unusual Bile Acids in Mice

“…Further histological analysis in the GI tract of these animals, particularly comparing pups to adults and with other staining methods, may reveal more abnormalities in the absorptive tissue of KO mice. Other physiological parameters compared between B aat KO and WT were initially performed by the animal’s source institution as part of the International Mouse Phenotyping Consortium ( 16 , 17 ) and are available online (see Materials and Methods ).…”

“…Cryo-preserved heterozygous sperm for strain C57BL/6NCrl- Baat em1(IMPC)Mbp /Mmucd (RRID:MMRRC_043495-UCD), originally created by Cas9 editing according to ( 16 , 17 ), was obtained from the Mutant Mouse Resource and Research Centers (MMRRC, University of California Davis). The genetic background of the animals is described on the MMRRC website ( https://www.mmrrc.org/catalog/sds.php?mmrrc_id=43495 ).…”

“…The genetic background of the animals is described on the MMRRC website ( https://www.mmrrc.org/catalog/sds.php?mmrrc_id=43495 ). Exon 2 ENSMUSE00000229562 and flanking splicing regions were constitutively deleted from the Baat gene ENSMUST00000043056.8 using CRISPR Cas9 gene editing technology in mouse zygotes ( 16 , 17 ); this deletion includes the highly conserved 328 Asp and 362 His in the active site ( 18 ). Cryo-recovery was performed via in-vitro fertilization at the Michigan State University Transgenic and Genome Editing Facility.…”

“…Animal numbers per sex, genotype, and experiment are shown in supplemental Table S1 . Further physiological characterization of B aat -/- animals was initially performed by the source laboratory ( 16 , 17 ) and is available online at: https://www.mousephenotype.org/data/genes/MGI:106642#phenotypes-section .…”

Comparative and longitudinal analysis of axial and retinal biometry in prospective models of hyperopia

“…To perform a detailed and comparative analysis of temporal changes in AL and retinal biometry, we selected aged Mfrp rd6 , Prss56 glcr4 and Adipor1 tm1Dgen murine models of genes which had previously been implicated in hyperopia and/or decreased AL [28,36,40] . In addition, we selected two uncharacterized JAX-KOMP 2 strains; Prss56 em2(IMPC)J , harboring a novel allele produced using Cas9-mediated genome editing [59] and C1qtnf5 tm1.1(KOMP)Vlcg , which harbors a reporter disruption in exons 2 and 3 of C1qtnf5 . Given that C1qtnf5 and Mfrp are expressed as dicistronic genes and are known to interact, we questioned whether this new knockout model would have phenotypic similarities with Mfrp rd6 .…”