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Easi-CRISPR: Efficient germline modification with long ssDNA donors

By Rolen M. Quadros, Masato Ohtsuka, Donald W Harms, Tomomi Aida, Ronald Redder, Hiromi Miura, Guy P Richardson, Mark A Behlke, Sarah A Zeiner, Ashley M Jacobi, Lisa D Urness, Suzanne L. Mansour, Channabasavaiah B. Gurumurthy

Posted 17 Aug 2016
bioRxiv DOI: 10.1101/069963 (published DOI: 10.1186/s13059-017-1220-4)

CRISPR/Cas9 technology efficiently produces short insertions or deletions (indels) and can insert short exogenous sequences at Cas9 cut sites. However, targeting long inserts is still a major technical challenge. To overcome this challenge, we developed Easi-CRISPR (Efficient additions with ssDNA inserts-CRISPR), a method that uses long, in vitro-synthesized, single-stranded DNAs with 50-100 base homology arms as repair templates. We demonstrate that Easi-CRISPR can generate knock-in and floxed alleles in mice with an efficiency at many loci as high as 100%. The simple design requirements for donor DNAs and the reproducibly high-efficiency of Easi-CRISPR enables rapid development of many types of commonly used animal and cell models.

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