Efficient generation of Rosa26 knock-in mice using CRISPR/Cas9 in C57BL/6 zygotes

Abstract

BackgroundThe CRISPR/Cas9 system is increasingly used for gene inactivation in mouse zygotes, but homology-directed mutagenesis and use of inbred embryos are less established. In particular, Rosa26 knock-in alleles for the insertion of transgenes in a genomic ‘safe harbor’ site, have not been produced. Here we applied CRISPR/Cas9 for the knock-in of 8–11 kb inserts into Rosa26 of C57BL/6 zygotes.ResultsWe found that 10–20 % of live pups derived from microinjected zygotes were founder mutants, without apparent off-target effects, and up to 50 % knock-in embryos were recovered upon coinjection of Cas9 mRNA and protein. Using this approach, we established a new mouse line for the Cre/loxP-dependent expression of Cas9.ConclusionsAltogether, our protocols and resources support the fast and direct generation of new Rosa26 knock-in alleles and of Cas9-mediated in vivo gene editing in the widely used C57BL/6 inbred strain.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0234-4) contains supplementary material, which is available to authorized users.

Efficient CRISPR-mediated mutagenesis in primary immune cells using CrispRGold and a C57BL/6 Cas9 transgenic mouse line

“…In the mice created by Platt et al (6) (hereafter called R26-Cas9p2aGFP), the Cas9 protein is fused to eGFP via a self-cleaving P2A peptide. In our Rosa26-LSL-Cas9iGFP mice, generated by CRISPRmediated editing of C57BL/6 zygotes, Cas9 is also expressed from the Rosa26 locus in a Cre-dependent manner, but with the Cas9 coding sequence linked to eGFP via an internal ribosomal entry site (IRES) (9). To apply the CRISPR/Cas9 technology to primary immune cells, we generated Cas9-transgenic mice with ubiquitous expression of Cas9 by crossing Rosa26-LSL-Cas9iGFP with Cre-deleter mice (Fig.…”

“…In addition, these mice were not generated on a pure C57BL/6 background. We thus generated a C57BL/6 Cas9-transgenic line with an improved Cas9 expression cassette (9). As we were interested in small-scale screening, a key consideration was to use only few, but reliable single guide RNAs (sgRNAs) without the necessity of sgRNA-testing experiments before screening.…”

Efficient generation of targeted large insertions in mouse embryos using 2C-HR-CRISPR

“…However, the efficiency of precise introduction of large fragments by homologous recombination (HR) is still not ideal 4,5 . Previous reports have suggested methods to increase large fragment knock-in efficiency in zygotes, by Cas9-RNP injection 6 , with templates activating micro-homology mediated end joining or long-homology mediated end joining pathways 4,7 , with long singlestranded DNA templates or by chemically manipulating DNA repair pathways 8,9 .…”

“…Some methods produce imprecise junctions at editing sites 7 , while others are limited by practical considerations and the size of the DNA fragment that can be inserted (up to 2 kb) 8 .…”

“…http://dx.doi.org/10.1101/204339 doi: bioRxiv preprint first posted online Oct. 16, 2017; 3 date 4 . Some methods produce imprecise junctions at editing sites 7 , while others are limited by practical considerations and the size of the DNA fragment that can be inserted (up to 2 kb) 8 . Fluorescent reporter mouse lines, which allow for direct visualization and live imaging of molecular and cellular dynamics during developmental, regenerative and pathological processes are among the most commonly generated models using large fragment knock-in.…”