Quadros, Rolen M, Miura, Hiromi, Harms, Donald W, Akatsuka, Hisako, Sato, Takehito, Aida, Tomomi, Redder, Ronald, Richardson, Guy P, Inagaki, Yutaka, Sakai, Daisuke, Buckley, Shannon M, Seshacharyulu, Parthasarathy, Batra, Surinder K, Behlke, Mark A, Zeiner, Sarah A, Jacobi, Ashley M, Izu, Yayoi, Thorenson, Wallace B, Urness, Lisa D, Mansour, Suzanne L, Ohtsuka, Masato and Gurumurthy, Channabasavaiah B (2017) Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins. Genome Biology, 18 (1). a92 1-15. ISSN 1474-760X

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Abstract

Background
Conditional knockout mice and transgenic mice expressing recombinases, reporters, and inducible transcriptional activators are key for many genetic studies and comprise over 90% of mouse models created. Conditional knockout mice are generated using labor-intensive methods of homologous recombination in embryonic stem cells and are available for only ~25% of all mouse genes. Transgenic mice generated by random genomic insertion approaches pose problems of unreliable expression, and thus there is a need for targeted-insertion models. Although CRISPR-based strategies were reported to create conditional and targeted-insertion alleles via one-step delivery of targeting components directly to zygotes, these strategies are quite inefficient.

Results
Here we describe Easi-CRISPR (Efficient additions with ssDNA inserts-CRISPR), a targeting strategy in which long single-stranded DNA donors are injected with pre-assembled crRNA + tracrRNA + Cas9 ribonucleoprotein (ctRNP) complexes into mouse zygotes. We show for over a dozen loci that Easi-CRISPR generates correctly targeted conditional and insertion alleles in 8.5–100% of the resulting live offspring.

Conclusions
Easi-CRISPR solves the major problem of animal genome engineering, namely the inefficiency of targeted DNA cassette insertion. The approach is robust, succeeding for all tested loci. It is versatile, generating both conditional and targeted insertion alleles. Finally, it is highly efficient, as treating an average of only 50 zygotes is sufficient to produce a correctly targeted allele in up to 100% of live offspring. Thus, Easi-CRISPR offers a comprehensive means of building large-scale Cre-LoxP animal resources.

Item Type:	Article
Keywords:	CRISPR/Cas9, Homology directed repair, Easi-CRISPR, long ssDNA donors, CRISPR ribonucleoproteins, Cre-LoxP, Conditional knockout, Reporter and recombinase knock-in
Schools and Departments:	School of Life Sciences > Neuroscience
Research Centres and Groups:	Sussex Neuroscience
Depositing User:	Guy Richardson
Date Deposited:	06 Dec 2017 09:37
Last Modified:	13 Aug 2020 10:15
URI:	http://sro.sussex.ac.uk/id/eprint/71794

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