Consistent Production of Mice with Conditional Knockout Alleles by CRISPR/Cas9-Mediated Genome Editing Using Two Guides/Two Oligos Approach
2020
Golovko, A. | Adams, J. | Guo, Huiping | Ballard, J. | Gonzales, A. | Morpurgo, B.
Gene targeting is extensively used to generate designer mouse mutants and to study gene function in vivo. Knockout mice that harbor a null allele in their germline provide appropriate genetic models of inherited diseases and often exhibit embryonic or early postnatal lethality. To study gene function in adult mice and in selected cell types, a refined strategy for conditional gene inactivation has been developed that relies on the DNA recombinase Cre and its recognition (loxP) sites. This process has traditionally relied on the complex process involving genome editing in embryonic stem (ES) cells despite its limitations, including incorrect targeting or cassette structure, and difficulties with germline transmission of the allele from chimeric mice. CRISPR-Cas9 gene editing technology has considerably facilitated the generation of mouse knockout alleles, relieving many of the cumbersome and time-consuming steps of traditional mouse ES cell technology. However, the generation of conditional knockout alleles remains an important challenge. An earlier study reported up to 16% efficiency in generating conditional knockout alleles in mice using 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides (ssODN), which has been questioned by another report combining data from multiple transgenic cores. With the advent of CRISPR/Cas9 as a mouse genome modification tool, we assessed the efficiency of using this method in creating conditional targeted alleles in three genes, phosphatase and actin regulator 1 (Phactr1), apolipoprotein A-I (ApoA1), and actin-related protein T2 (Actrt2). Even though overall success rate was low—about 2.5%—we show that it’s possible to reliably generate conditional knockout alleles using CRISPR/Cas9 on a consistent basis.
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