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Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins

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posted on 2023-06-09, 09:10 authored by Rolen M Quadros, Hiromi Miura, Donald W Harms, Hisako Akatsuka, Takehito Sato, Tomomi Aida, Ronald Redder, Guy Richardson, Yutaka Inagaki, Daisuke Sakai, Shannon M Buckley, Parthasarathy Seshacharyulu, Surinder K Batra, Mark A Behlke, Sarah A Zeiner, Ashley M Jacobi, Yayoi Izu, Wallace B Thorenson, Lisa D Urness, Suzanne L Mansour, Masato Ohtsuka, Channabasavaiah B Gurumurthy
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.

Funding

The Tectorial Membrane and the Sensory Hair Bundles of the Inner Ear: Mechanisms of Development and Effects of Deafness-Causing Mutations; G0162; WELLCOME TRUST; 087737

History

Publication status

  • Published

File Version

  • Published version

Journal

Genome Biology

ISSN

1474-760X

Publisher

BioMed Central

Issue

1

Volume

18

Page range

1-15

Article number

a92

Department affiliated with

  • Neuroscience Publications

Research groups affiliated with

  • Sussex Neuroscience Publications

Full text available

  • Yes

Peer reviewed?

  • Yes

Legacy Posted Date

2017-12-06

First Open Access (FOA) Date

2017-12-06

First Compliant Deposit (FCD) Date

2017-12-06

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