Abstract
The CRISPR/Cas9 tool can easily generate knockout mouse models by disrupting the gene sequence, but its efficiency for creating models that require either insertion of exogenous DNA (knock-in) or replacement of genomic segments is very poor. The majority of mouse models used in research are knock-in (reporters or recombinases) or gene-replacement (for example, conditional knockout alleles containing LoxP sites flanked exons). A few methods for creating such models are reported using double-stranded DNA as donors, but their efficiency is typically 1–10% and therefore not suitable for routine use. We recently demonstrated that long single-stranded DNAs serve as very efficient donors, both for insertion and for gene replacement. We call this method Easi-CRISPR (efficient additions with ssDNA inserts-CRISPR), a highly efficient technology (typically 25%-50%, and up to 100% in some cases), one that has worked at over a dozen loci thus far. Here, we provide detailed protocols for Easi-CRISPR.
