241 CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS (CRISPR)/Cas9 ASSOCIATED WITH TRANSIENT DEPLETION OF NON-HOMOLOGOUS END-JOINING PATHWAY INCREASED GENE‐TARGETING EFFICIENCY IN GOAT FIBROBLASTS

Abstract

The transgenic animal platform for the expression of recombinant proteins in the milk offers particularly attractive possibilities. The recent application of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 to promote precise genome modifications and DNA editing also allows the targeting of specific DNA sequences in embryos or cells in culture. In addition, the transient knockdown of the NHEJ pathway by RNAi has been shown to increase gene‐targeting (GT) rate in cultured cells (Bertolini et al. 2009 Mol. Biotechnol. 41, 106–114). The CRISPR/Cas9 system was used to target a 15-kb transgene construct into the ROSA26 locus in goat fetal fibroblast cells subjected to a transient RNAi‐induced depletion of the NHEJ Ku70 protein. A polycistronic expression vector was constructed by ligating the coding sequences of 3 antigenic proteins against Brucella abortus linked by self-processing 2A peptides under the regulation of the bovine α-lactalbumin promoter. The final vector also contained the neomycin resistance gene and left and right 2-kb arms homologous to the goat ROSA26 locus. A total of 2 × 10⁵ fibroblast cells at passage 3 from a 50-day fetus were transfected using the Neon Transfection System (Invitrogen/ThermoFisher Scientific, Waltham, MA, USA), according to the following groups: mock control (M); vector-only (V); vector + RNAi against Ku70 (VR); vector + ROSA26‐CRISPR/Cas9 (VC); and vector + RNAi against Ku70 + ROSA26-CRISPR/Cas9 (VCR). After antibiotic selection, colonies were characterised for zygosity, transgene copy number, and off-targets. Mortality rates following cell transfection were 68, 78, 75, 83, and 90%, and the number of colonies after selection was 0, 13, 22, 5, and 8 for the M, V, VR, VC, and VCR groups, respectively. Gene targeting was detected only when the ROSA26‐CRISPR/Cas9 was combined to the vector (VC group, 1 in 22 colonies) or to the vector and RNAi against Ku70 (VCR group, 1 in 8 colonies), with a 2.8-fold increase in GT rate when associating the 3 components (VCR group). No CRISPR/Cas9 off-targets were detected in 7 different sequenced hot spots. One colony from the VC group, harboring a biallelic transgene knock-in, was chosen for use in goat cloning by SCNT following our established procedures (Martins et al. 2015 Reprod. Fertil. Dev. 27, 111). Four viable pregnancies (33.3%) were established, based on the ultrasonographic visualisation of the embryo and heartbeat on Day 26, after the transfer of 144 embryos to 12 female recipient does, demonstrating the developmental potential of the transgenic knock-in donor cells. However, pregnancies were lost up to Day 55 of pregnancy. Our preliminary data suggest that the combined cell transfection of gene target-specific CRISPR/Cas9 and RNAi to knockdown the NHEJ pathway is a viable and efficient approach to produce precise genetically modified goat donor cells carrying mono- and biallelic knock-ins of large size transgene constructs for use in cloning by SCNT. Cloning procedures are underway using biallelic knock-in somatic cells to obtain live offspring, which will be the first step to produce and test a recombinant subunit vaccine against B. abortus.