Cre-dependent Cas9-expressing pigs enable efficient in vivo genome editing

Abstract

Despite being time-consuming and costly, generating genome-edited pigs holds great promise for agricultural, biomedical, and pharmaceutical applications. To further facilitate genome editing in pigs, we report here establishment of a pig line with Cre-inducible Cas9 expression that allows a variety of ex vivo genome editing in fibroblast cells including single- and multigene modifications, chromosome rearrangements, and efficient in vivo genetic modifications. As a proof of principle, we were able to simultaneously inactivate five tumor suppressor genes (, ,, , and) and activate one oncogene (), achieved by delivering Cre recombinase and sgRNAs, which caused rapid lung tumor development. The efficient genome editing shown here demonstrates that these pigs can serve as a powerful tool for dissecting in vivo gene functions and biological processes in a temporal manner and for streamlining the production of genome-edited pigs for disease modeling.

Platforms of in vivo genome editing with inducible Cas9 for advanced cancer modeling

“…One approach is to externally deliver CRISPR/Cas9 directly into the animal's body by various methods such as virus and electroporation; however, low mutation efficiency in the target organ and variation in the resultant mutants may preclude the use of this system for in vivo experiments. Another approach is Cas9 ‐transgenic animals, in which either Cre/loxP‐conditional or Dox‐inducible Cas9 are used to alleviate the challenges of delivery. Especially in the latter report, sgRNAs were targeted into the genome together with an inducible Cas9 so that the mice were germline‐competent with improved mutation efficiency and homogeneity.…”

A Novel Cre Recombinase-Mediated In Vivo Minicircle DNA (CRIM) Vaccine Provides Partial Protection against Newcastle Disease Virus

“…To achieve this goal, some inducible promoters, such as the arabinosedependent araC P BAD promoter (18) and temperature-sensitive cI857/PR promoter (15), have been used previously for efficient production of mcDNA in vitro. Another approach for regulated synthesis of recombinase usually relies on in vivo promoters such as the cytomegalovirus (CMV) promoter (34)(35)(36) and phosphoglycerate kinase (PGK) promoter (37). Different transcription and translation efficiencies for the CMV and PGK promoters have been previously reported because the CMV promoter could also significantly initiate gene transcription in Escherichia coli strains (38), which compromises the application of this promoter for our purpose, i.e., for strict control of the expression of recombinase in vitro.…”

Resources for genome editing in livestock: Cas9-expressing chickens and pigs

“…Moreover, it is readily targeted, supports abundant ubiquitous expression, and is dispensable for normal physiology and development (28, 29). Unlike previous work, where a Cre-inducible Cas9-expressing line has been generated (36), our pig line expresses a single copy of SpCas9 ubiquitously under the control of the CBh promoter. This eliminates the need for two consecutive recombination events to occur and should increase editing efficiency.…”

“…This eliminates the need for two consecutive recombination events to occur and should increase editing efficiency. For example, only 0.1% of cells expressed both Cre-recombinase and the inducible Cas9 after in vivo lentiviral-mediated delivery of Cre (36). Ubiquitously Cas9-expressing pigs are healthy and fertile and could be bred to homozygosity, which agrees with findings made in ROSA26 -Cas9-expressing mice (27).…”