TIGER: Single-step in vivo genome editing in a non-traditional rodent

Abstract

Rodents are taxonomically diverse and have evolved a variety of traits. A mechanistic understanding of such traits has remained elusive, however, largely because genome editing in non-traditional model species remains challenging. Here, using the African striped mouse (Rhabdomys pumilio), we describe TIGER (targeted in vivo genome editing in rodents), a method that relies on a simple intraoviductal injecting technique and uses recombinant adeno-associated viruses (rAAVs) as the sole vehicle to deliver reagents into pregnant females. We demonstrate that TIGER generates knockout and knockin (up to 3 kb) lines with high efficiency. Moreover, we engineer a double-cleaving repair rAAV template and find that it significantly increases knockin frequency and germline transmission rates. Lastly, we show that an oversized double-cleaving rAAV template leads to an insertion of 3.8 kb. Thus, TIGER constitutes an attractive alternative to traditional ex vivo genome-editing methods and has the potential to be extended to a broad range of species.

Keywords: African striped mice; CP: Developmental biology; CP: Molecular biology; CRISPR; CRISPR knock-in; CRISPR knock-out; Rhabdomys pumilio; emerging model organisms; intraoviductal injections; in vivo genome editing; non-traditional rodents.