Rapid production of precisely edited cattle and mice via a single AAV delivered miniature IscB#/?RNA# [dataset 1]

Utilising adeno-associated virus (AAV) to deliver CRISPR/Cas has enhanced the generation of knock-out mice, but single-AAV delivery of Cas and homology-directed-repair (HDR) templates in livestock remains a challenge. Here, we introduce all-in-one AAV vectors containing shortened IscB#, the progenitor of Cas9, along with either miniature ?RNA#, or ?RNA# with a large HDR-template. Encouragingly, we efficiently generated knock-out and knock-in cattle and mice by infecting zygotes with AAV::IscB#/?RNA#-HDR without costly micromanipulators. Overall design: The novel strategy to generate genetically modified mice and ruminant cattle by transduction of engineered IscB and its ?RNA (termed IscB#-?RNA#) components into pre-implantation embryos via co-culture with adeno-associated viruses (AAVs).