Combination of TALEN and HITI gene edition technologies for KI of recombinant human factor IX (rhFIX) under βCasein (CSN2) native promoter in bovine IVF embryos

BEVACQUA RJ; RATNER LD; TABOGA OA; FAHRENKRUG SC; CANEL N; CARLSON DF; FERRARIS S; SALAMONE DF; SAVY V; ALBERIO V; GISMONDI MI; RULLI SB; FERNANDEZ Y MARTIN RAFAEL

New Orleans

Conferencia; 45th Annual Conference of the IETS; 2019

International Embryos Technology Society

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, transcriptionactivator-like effector nucleases (TALEN), and CRISPR/Cas9. Still, precise knock-in (KI) techniques remain inefficient. Recently, the homologyindependent target integration (HITI) strategy was developed, allowing precise insertion of transgenes in mammalian cells in an easier fashion. TheHITI technique allows site-specific gene insertion by means of cleavage of both the target sequence in the genome and the donor plasmid, followed byDNA repair by nonhomologous end joining. Here, we evaluated the use of TALENs to generate precise knockout (KO) alleles of the b-casein gene(CSN2) by creating small insertions or deletions, and precise insertion of recombinant human factor IX (rhFIX) under bovine CSN2 regulatorysequences, using HITI via cytoplasmic injection of bovine IVF zygotes. First, 2 TALEN pairs (Tn1 and Tn2) targeting exon 2 of bovine CSN2 weredesigned and their activity was confirmed by primary fibroblasts transfection followed by Surveyor assay at Day 3. Then, both TALEN pairs wereevaluated for KO embryo generation by zygote cytoplasmic injection of in vitro-transcribed mRNA encoding for Tn1, Tn2, or a mix containingTn1þTn2, at 100 ng mL1. A non-injected control (NIC) was also included. Embryos were in vitro cultured until Day 7 and independently analysed bywhole-genome amplification followed by PCR and sequencing. Neither the blastocyst rate [28.8% (n ¼ 73), 33.8% (n ¼ 71), 32.4% (n ¼ 74), and54.3% (n ¼ 127) for Tn1, Tn2, Tn1þTn2, and NIC, respectively] nor the proportion of edited embryos [44% (n ¼ 9), 20% (n ¼ 10), and 33% (n ¼ 9)for Tn1, Tn2, and Tn1þTn2, respectively] differed between injected groups (Fisher test, P , 0.05), demonstrating efficient editing in bovine embryosby TALENs. Finally, to achieve precise CSN2 KI embryos, the rhFIX open reading frame was PCR amplified with a forward primer containing theTn1 recognition sequence to obtain the HITI donor and bovine IVF zygotes were co-injected with the Tn1 mRNA and the HITI donor. Embryos werein vitro cultured until Day 7 and individually analysed by nested PCR at both the 50 and 30 ends of HITI donor. The PCR-based results indicate HITIdonor integration in 7% of embryos analysed (n ¼ 14). Sanger sequencing analysis is currently in progress to confirm site-specific integration of HITIand possible rearranged DNA integration in other embryos. To our knowledge, this is the first report on the use of TALEN and HITI for genemodification. Our results indicate that TALEN combined with HITI may constitute an easy strategy for precise production of pharmaceuticals in themilk of livestock.