Easi-CRISPR: Efficient germline modification with long ssDNA donors

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 typ