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

V SAVY, RJ BEVACQUA, NG CANEL, V ALBERIO, LD RATNER, DF CARLSON, MI GISMONDI, O TABOGA, S FERRARIS, SB RULLI, SC FAHRENKRUG, DF SALAMONE, R FERNANDEZ-MARTÍN

new orleans

Congreso; International Embryo Technology Society?s 45th Annual Meeting; 2019

International Embryo 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, transcription activator-like effector nucleases (TALEN) and CRISPR/Cas9. Still, precise Knock in (KI) techniques remain inefficient. Recently, Homology-independent target integration strategy (HITI) was developed, allowing precise insertion of transgenes in mammalian cells in an easier fashion. Here we evaluated the use of TALEN to generate precise KO alleles of CSN2, and TALEN combined with HITI for the precise insertion of rhFIX under bovine CSN2 regulatory sequences by cytoplasmic injection of bovine IVF zygotes, as a potential strategy for low-cost production of pharmaceuticals in bovine milk.First, two 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 with a Tn1+Tn2 mix, at 100 ng/μl. A non-injected control (NIC) was also included. Embryos were in vitro cultured until day 7 and independently analyzed by whole-genome amplification followed by PCR and sequencing. Neither the blastocyst rates [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), 33% (n=9) for Tn1, Tn2 and Tn1+Tn2 respectively] differed between injected groups (p