In situ genome editing method suitable for routine generation of germline modified animal models

Abstract

Animal genome engineering experimental procedures involve three major steps: isolation of zygotes from pregnant females; microinjection of zygotes, and; transfer of injected zygotes into recipient females, that have been practiced for over three decades. The laboratory set ups intending to performing these procedures require to have sophisticated equipment as well as highly skilled technical personnel. Because of these reasons, animal transgenesis experiments are typically performed at centralized core facilities in most research organizations. We recently showed that all three steps, of animal transgensis, can be by-passed using a method termed GONAD (Genome-editing via Oviductal Nucleic Acids Delivery), by directly electroporating genome editing components into zygotes in situ . Although our first report demonstrated the genome-editing capability, its efficiency was lower than the standard methods using microinjection. Here we investigated critical parameters of GONAD to make it suitable for creating animal models of large genomic deletions, single nucleotide corrections and long sequence insertions. The efficiency of genome editing in the improved GONAD ( i -GONAD) method reached to the levels comparable to traditional microinjection methods. The streamlined parameters, and the simplified experimental steps, in the i -GONAD method makes it suitable for routine genome editing applications performed both at centralized facilities as well as at the laboratories that lack highly skilled personnel and the sophisticated equipment.