202 Combination of transcription activator-like effector nucleases and homology-independent target integration strategy gene editing technologies for knock-in of recombinant human factor IX Under the β-casein native promoter in bovine IVF embryos

Abstract

Precise DNA modification is a crucial approach for gene function elucidation, biomedical model development, and transgenic bioreactor generation. In livestock, its application was extremely challenging until the development of engineered nucleases such as zinc-finger nucleases, transcription activator-like effector nucleases (TALEN), and CRISPR/Cas9. Still, precise knock-in (KI) techniques remain inefficient. Recently, the homology-independent target integration (HITI) strategy was developed, allowing precise insertion of transgenes in mammalian cells in an easier fashion. The HITI technique allows site-specific gene insertion by means of cleavage of both the target sequence in the genome and the donor plasmid, followed by DNA repair by nonhomologous end joining. Here, we evaluated the use of TALENs to generate precise knockout (KO) alleles of the β-casein gene (CSN2) by creating small insertions or deletions, and precise insertion of recombinant human factor IX (rhFIX) under bovine CSN2 regulatory sequences, using HITI via cytoplasmic injection of bovine IVF zygotes. First, 2 TALEN pairs (Tn1 and Tn2) targeting exon 2 of bovine CSN2 were designed and their activity was confirmed by primary fibroblasts transfection followed by Surveyor assay at Day 3. Then, both TALEN pairs were evaluated for KO embryo generation by zygote cytoplasmic injection of in vitro-transcribed mRNA encoding for Tn1, Tn2, or a mix containing Tn1+Tn2, at 100 ng μL⁻¹. A non-injected control (NIC) was also included. Embryos were in vitro cultured until Day 7 and independently analysed by whole-genome amplification followed by PCR and sequencing. Neither the blastocyst rate [28.8% (n = 73), 33.8% (n = 71), 32.4% (n = 74), and 54.3% (n = 127) for Tn1, Tn2, Tn1+Tn2, and NIC, respectively] nor the proportion of edited embryos [44% (n = 9), 20% (n = 10), and 33% (n = 9) for Tn1, Tn2, and Tn1+Tn2, respectively] differed between injected groups (Fisher test, P < 0.05), demonstrating efficient editing in bovine embryos by TALENs. Finally, to achieve precise CSN2 KI embryos, the rhFIX open reading frame was PCR amplified with a forward primer containing the Tn1 recognition sequence to obtain the HITI donor and bovine IVF zygotes were co-injected with the Tn1 mRNA and the HITI donor. Embryos were in vitro cultured until Day 7 and individually analysed by nested PCR at both the 5′ and 3′ ends of HITI donor. The PCR-based results indicate HITI donor integration in 7% of embryos analysed (n = 14). Sanger sequencing analysis is currently in progress to confirm site-specific integration of HITI and possible rearranged DNA integration in other embryos. To our knowledge, this is the first report on the use of TALEN and HITI for gene modification. Our results indicate that TALEN combined with HITI may constitute an easy strategy for precise production of pharmaceuticals in the milk of livestock.