Reproducibility of CRISPR-Cas9 methods for generation of conditional mouse alleles: a multi-center evaluation

Abstract

Background CRISPR-Cas9 gene-editing technology has facilitated the generation of knockout mice, providing an alternative to cumbersome and time-consuming traditional embryonic stem cell-based methods. An earlier study reported up to 16% efficiency in generating conditional knockout (cKO or floxed) alleles by microinjection of 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides as donors (referred herein as “two-donor floxing” method). Results We re-evaluate the two-donor method from a consortium of 20 laboratories across the world. The dataset constitutes 56 genetic loci, 17,887 zygotes, and 1718 live-born mice, of which only 15 (0.87%) mice contain cKO alleles. We subject the dataset to statistical analyses and a machine learning algorithm, which reveals that none of the factors analyzed was predictive for the success of this method. We test some of the newer methods that use one-donor DNA on 18 loci for which the two-donor approach failed to produce cKO alleles. We find that the one-donor methods are 10- to 20-fold more efficient than the two-donor approach. Conclusion We propose that the two-donor method lacks efficiency because it relies on two simultaneous recombination events in cis , an outcome that is dwarfed by pervasive accompanying undesired editing events. The methods that use one-donor DNA are fairly efficient as they rely on only one recombination event, and the probability of correct insertion of the donor cassette without unanticipated mutational events is much higher. Therefore, one-donor methods offer higher efficiencies for the routine generation of cKO animal models. Electronic supplementary material The online version of this article (10.1186/s13059-019-1776-2) contains supplementary material, which is available to authorized users.

Genes adapt to outsmart gene-targeting strategies in mutant mouse strains by skipping exons to reinitiate transcription and translation

“…Systematic generation and phenotyping of thousands of KO mouse strains by the IMPC is one of the most significant biological resources available to researchers for understanding mammalian gene function [ 37 ]. Despite advances in CRISPR-Cas genome editing technologies, generating conditional KOs is rather challenging [ 38 ], and furthermore, targeted KO-first alleles offer a unique advantage by facilitating monitoring of gene function in a tissue- and temporal-specific manner. Nevertheless, the present study emphasizes that nature can circumvent gene-targeting strategies to affect the phenotype of homozygous mutant mice.…”

LRRC8A is essential for hypotonicity-, but not for DAMP-induced NLRP3 inflammasome activation

“…Only two of these exons contain coding sequence, with exon three harbouring >85% of the coding sequence and possessing large introns, and thus an ideal candidate for floxing. We initially attempted the 2-sgRNA, 2-oligo approach described previously ( Yang et al, 2013 ), but failed to obtain mice with both loxP integrated on the same allele ( Gurumurthy et al, 2019 ). Instead, a colony from a single founder with the 5’ LoxP integrated was established, bred to homozygosity, and used as a background to integrate the second 3’ loxP.…”

Embryo-Based Large Fragment Knock-in in Mammals: Why, How and What’s Next

“…This was achieved by microinjecting Cas9 mRNA, an sgRNA, and a plasmid-based homology template into the cytoplasm of newly fertilized zygotes. However, the efficiency of this particular technology has since been shown to be highly variable and dependent on genetic locus [30,59]. Since then, several technological modifications have been developed that have led to reported knock-in efficiencies of up to 95%-100% in some gene loci.…”