Generation of CRISPR/Cas9-mediated gene-targeted pigs via somatic cell nuclear transfer

Abstract

The domestic pig has been widely used as an important large animal model. Precise and efficient genetic modification in pig provides a great promise in biomedical research. Recently, clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system has been successfully used to produce many gene-targeted animals. However, these animals have been generated by co-injection of Cas9 mRNA and single-guide RNA (sgRNA) into one-cell stage embryos, which mostly resulted in mosaicism of the modification. One or two rounds of further breeding should be performed to obtain homozygotes with identical genotype and phenotype. To address this issue, gene-targeted somatic cells can be used as donor for somatic cell nuclear transfer (SCNT) to produce gene-targeted animals with single and identical mutations. In this study, we applied Cas9/sgRNAs to effectively direct gene editing in porcine fetal fibroblasts and then mutant cell colonies were used as donor to generate homozygous gene-targeted pigs through single round of SCNT. As a result, we successfully obtained 15 tyrosinase (TYR) biallelic mutant pigs and 20 PARK2 and PINK1 double-gene knockout (KO) pigs. They were all homozygous and no off-target mutagenesis was detected by comprehensive analysis. TYR (-/-) pigs showed typical albinism and the expression of parkin and PINK1 were depleted in PARK2 (-/-)/PINK1 (-/-) pigs. The results demonstrated that single- or double-gene targeted pigs can be effectively achieved by using the CRISPR/Cas9 system combined with SCNT without mosaic mutation and detectable off-target effects. This gene-editing system provides an efficient, rapid, and less costly manner to generate genetically modified pigs or other large animals.

Exogenous enzymes upgrade transgenesis and genetic engineering of farm animals

“…Despite the demonstration that CRSPR/Cas9 works in livestock cells, recovery of CRISPR mutant cell clones was much lower than that with TALENs, suggesting that CRISPR/Cas9 system needs further optimization to achieve targeting efficiencies comparable to TALENs. Follow-up studies have demonstrated that CRISPR/Cas9 genomeedited cells can support development to term when used as nuclear donor in SCNT in pigs [244][245][246] and goats [247].…”

Insert, remove or replace: A highly advanced genome editing system using CRISPR/Cas9

“…One-step generation of mutants, which means that biallelic targeting with efficiency close to 100 % is achieved, has been published in pigs, for example, for the von Willebrand Factor gene [67] using zygote injections of sgRNAs and Cas9. CRISPR/Cas9 targeting in cell lines followed by SCNT can also reduce the need for breeding as it has been exemplified by generation of PARK2 -/-PINK1 -/-double mutant for modeling Parkinson"s disease [68]. Multiple knock-in strategies have also been developed in the pig, one interesting example of which is Oct4-tdTomato pig transgenic reporter line produced using SCNT, where expression of tdTomato was present in pluripotent cells of the embryos [69].…”

Potential alternative approaches to xenotransplantation

“…In addition, they demonstrated that blastocyst complementation is reproducible in pigs by using somatic cell nuclear transfer (cloning) technology [51]. New emerging genome-modifying technology, e.g., TALENs or the CRISPR/Cas9 system, has made the construction of organ-deficient pigs simpler and more convenient [52][53][54]. although several options have been proposed to resolve such a concern [56].…”