Precision exogenous gene knock-in is an attractive field for transgenic Gallus gallus (chicken) generation. In this article, we constructed multiple Precise Integration into Target Chromosome (PITCh) plasmid systems mediated by microhomology-mediated end-joining (MMEJ) for large-fragment integration in DF-1 cells and further assess the possibility of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as a genomic safe harbor for chickens. We designed three targeted sgRNAs for the all-in-one plasmid at the 3'UTR of GAPDH near the stop codon. The donor-plasmid-carrying microhomology arms correspond to sgRNA and EGFP fragments in the forward and reverse directions. MMEJ-mediated EGFP insertion can be efficiently expressed in DF-1 cells. Moreover, the differences between the forward and reverse fragments indicated that promoter interference does affect the transfection efficiency of plasmids and cell proliferation. The comparison of the 20 bp and 40 bp microhomology arms declared that the short one has higher knock-in efficiency. Even though all three different transgene insertion sites in GAPDH could be used to integrate the foreign gene, we noticed that the G2-20R-EGFP cell reduced the expression of GAPDH, and the G3-20R-EGFP cell exhibited significant growth retardation. Taken together, G1, located at the 3'UTR of GAPDH on the outer side of the last base of the terminator, can be a candidate genomic safe harbor (GSH) loci for the chicken genome. In addition, deleted-in-azoospermia-like (DAZL) and actin beta (ACTB) site-specific gene knock-in indicated that MMEJ has broad applicability and high-precision knock-in efficiency for genetically engineered chickens.
Keywords: CRISPR/Cas9; DF-1 cell line; GAPDH; MMEJ; precise integration; safe harbor loci.