Efficient Targeting Of FATS At AT-rich Sequence In Mice Through TALEN-mediated Double-hit Genome Modification

ObjectiveTranscription activator-like effector nucleases (TALENs) have emerged as a newly developed approach for genome editing and animal model generation since2011. However, its application to targeting specific genomic loci susceptible to DNA damage remains obscure. Here, we report a modified TALENs approach for efficient targeting FATS, a fragile-site gene with AT-rich sequence and di-nucleotide repeats in major introns. Two pairs of TALENs were designed to introduce targeted double stand breaks (DSBs) within a coding exon of FATS gene, in an effort to establish FATS knockout mice.Methods1. Bioinformatics methods were used to analyze the DNA sequence of mouse FATS gene and design the targeting sites of TALEN vectors. To improve the efficiency and activity of TALEN-mediated gene targeting, two TALEN-target sites at the largest exon coding the N-terminal FATS were selected.2."Golden Gate" method was used to assemble TALEN target vectors. The plasmid DNA was purified after verification by DNA sequencing.3. The FATS-TALEN vectors were electroporated into mouse HC11cells to check their cleavage efficiency in vitro.4. Having demonstrated the activity of FATS-TALEN in vitro, each FATS-TALEN vector was linearized and subjected to in vitro transcription. FATS-TALEN mRNAs were microinjected into the cytoplasm of pro-nuclear stage mouse embryos to produce offspring.5. Genotyping for the founder mice and test the specificity of genome editing by FATS-TALEN pairs.6. The founder mouse was crossed with a wild-type mouse to genetate F1offspring. Germline transmission of a FATS mutation was further tested using F1mice.7. The protein expression of FATS in knockout mice was examined by Western blot. Results1. Both of the two TALEN-FATS pairs exhibited cleavage activity in vitro, and the results of TA cloning and DNA sequencing verified their in vitro targeting efficiency as25%and4.5%, respectively.2. The in vivo targeting efficiency of two TALEN-FATS pairs was17.2%and14.3%, respectively. A three-to four-fold increase in targeting efficiency in mice was observed when two pairs of FATS-TALENs were simultaneously injected into one-cell mouse zygotes.3. The results of T7EN1and DNA sequencing confirmed that off-target effect of TALEN was not observed, demonstrating the targeting specificity of FATS-TALEN pairs.4. Germline transmission of FATS mutations was observed.5. The results of western blot verified the downregulation and silence of FATS protein expression in heterozygous and homozygous FATS-knockout mice, respectively.Conclusion1. Using the same concentration of FATS-TALEN pairs, the targeting efficiency in mice mediated by two pairs of FATS-TALEN is significantly higher than that mediated by one pair of FATS-TALEN.2. The specificity of TALEN-mediated gene targeting is confirmed, and mutations in founders are heritable through germline transmission. Therefore, genome editing using TALEN-mediated double-hit strategy exhibits higher efficiency of gene targeting, in comparison with single pair TALEN approach, to extablish gene knockout models, which will facilitate the further systematic study on the functions of fragile-site genes.