220 LENGTH OF DONOR DNA HOMOLOGY TO FACILITATE BI-ALLELIC GENE TARGETING DURING TRANSCRIPTION ACTIVATOR-LIKE EFFECTOR NUCLEASE-MEDIATED GENE TARGETING

Abstract

Gene editing techniques are increasing in their availability and ease of use. These techniques are an attractive alternative to conventional gene targeting methods via homologous recombination due to the ease of use and the efficiency of targeting. We have previously produced CMP-N-acetylneuraminic acid hydroxylase (CMAH) knockout (KO) pigs in a Minnesota miniature pig genetic background. These pigs were generated using zinc-finger nucleases (ZFN) in combination with donor DNA containing a total length of homology of 1600 bp (800-bp homology on each arm). A second KO was desired in the CMAH –/– genetic background, GGTA1. We used this opportunity to evaluate the effect of total donor homology on transcription activator-like effector nuclease (TALEN) mediated bi-allelic modification. Donor DNA was designed to symmetrically flank the predicted TALEN cleavage site in GGTA1 and was varied in length of total homology (40, 100, 200, 460, 890, and 1800 bp). The TALEN constructs and donor DNA were introduced into fibroblast cells by a lipid-based transfection method. Each donor DNA was co-transfected with a pair of TALEN constructs. In addition, the TALEN pair alone and the longest donor DNA alone were separately transfected to evaluate error-prone non-homologous end joining (NHEJ) repair or background HR, respectively. Five days post transfection, cells were evaluated for the ablation of the α-1,3 gal epitope, which results from bi-allelic disruption of GGTA1. Lectin IB-4 (FITC labeled) was used to detect the presence/absence of the epitope. If a bi-allelic modification to the GGTA1 gene was accomplished, specific binding of the lectin would not occur; therefore, a lack of FITC was evaluated. Donor DNA alone did not result in detectable bi-allelic conversion of GGTA1. As the length of donor DNA increased, the bi-allelic disruption of GGTA1 increased from 0.5% (no donor DNA) to a maximum of 3% (1800 bp). Inclusion of homologous donor DNA in TALEN transfections facilitates HR that competes with error-free NHEJ. The use of donor DNA with TALEN therefore can provide for increased bi-allelic disruption and predictability in the sequence of the resulting mutation.