In situgenome 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 bypassed 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.

Easi-CRISPR for creating knock-in and conditional knockout mouse models using long ssDNA donors

“…If necessary, silent mutations in the donor design can also be included to avoid re-cleavage. If suitable guides are not available for a locus, for targeting via Cas9, targeting via other CRISPR enzymes such as Cpf1 can also be considered[16].…”

“…The Easi- CRISPR method was developed using microinjection delivery in mice. The approach can also be adapted for use with electroporation or hydrodynamics gene delivery[14–16]. Easi- CRISPR can also be used to generate genome-edited animals in other species wherever zygote/embryo delivery of CRISPR components via microinjection or electroporation is possible (such as fruit flies[17], zebrafish[18], rat[19], and rabbit[20], as well as livestock species such as cattle[21], sheep[22], goats[23] and pigs[24]).…”