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bioRxiv · 10.1101/069963

Easi-CRISPR: Efficient germline modification with long ssDNA donors

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
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Abstract

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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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. 2016-08-17. Easi-CRISPR: Efficient germline modification with long ssDNA donors. https://doi.org/10.1101/069963

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