CRISPR-READI: Efficient Generation of Knockin Mice by CRISPR RNP Electroporation and AAV Donor Infection.
Cell Rep. 2019 06 25;27(13):3780-3789.e4
Authors: Chen S, Sun S, Moonen D, Lee C, Lee AY, Schaffer DV, He L
Genetically engineered mouse models harboring large sequence insertions or modifications are critical for a wide range of applications including endogenous gene tagging, conditional knockout, site-specific transgene insertion, and gene replacement; however, existing methods to generate such animals remain laborious and costly. To address this, we developed an approach called CRISPR-READI (CRISPR RNP electroporation and AAV donor infection), combining adeno-associated virus (AAV)-mediated HDR donor delivery with Cas9/sgRNA RNP electroporation to engineer large site-specific modifications in the mouse genome with high efficiency and throughput. We successfully targeted a 774 bp fluorescent reporter, a 2.1 kb CreERT2 driver, and a 3.3 kb expression cassette into endogenous loci in both embryos and live mice. CRISPR-READI is applicable to most widely used knockin schemes requiring donor lengths within the 4.9 kb AAV packaging capacity. Altogether, CRISPR-READI is an efficient, high-throughput, microinjection-free approach for sophisticated mouse genome engineering with potential applications in other mammalian species.
PMID: 31242412 [PubMed – indexed for MEDLINE]