Comparison of gene editing efficiencies of CRISPR/Cas9 and TALEN for generation of MSTN knock-out cashmere goats

• Published in: Theriogenology 2019-07, Vol.132, p.1-11
• Main Authors: Zhang, Ju, Liu, Jian, Yang, Wenliang, Cui, MengLan, Dai, Bai, Dong, Yanhua, Yang, Jie, Zhang, Xiaomeng, Liu, Dongjun, Liang, Hao, Cang, Ming
• Format: Article
• Language: English
• Subjects: CRISPR/Cas9; Goat; Knockout; Myostatin (MSTN); Somatic cell nuclear transfer; TALEN
• ISSN: 0093-691X; 1879-3231
• DOI: 10.1016/j.theriogenology.2019.03.029

The genome editors CRISPR/Cas9 (clustered regularly interspaced short palindromicrepeats/Cas9 nuclease-null) and TALENs (transcription activator-like effector nuclease) are popularly used for targeted modification of the mammalian genome. To date, few comparative studies have been carried out to investigate the differences between the use of CRISPR/Cas9 and TALENs in genome editing for goat breeding. Here, we compared CRISPR/Cas9 and TALEN technologies at multiple levels for generating a knock out (KO) of the Alpas cashmere goat myostatin (MSTN) gene, which negatively regulates the proliferation and differentiation of skeletal muscle cells. The electrotransfection efficiency observed using CRISPR/Cas9 was 8.1% more than that observed using TALEN for generating MSTN KO cells. In addition, the cutting efficiency of CRISPR/Cas9 for editing exon 1 of the MSTN gene was higher than that of TALENs. However, the off-target effects of the CRISPR/Cas9 system were also higher than those of TALENs. Further, we found that the frequency of obtaining MSTN−/- mutations by CRISPR/Cas9 was 8.5 times higher than that by TALEN. The CRISPR/Cas9-edited colonies involved longer deletions (up to 117 bp) than the TALEN-edited colonies (up to 13 bp). Remarkably, when embryos used to generate cloned goat via somatic cell nuclear transfer were compared, we found that the TALEN MSTN KO embryos easily developed to 8 cells and their cleavage rate was significantly higher than that of CRISPR/Cas9-edited embryos. Finally, we produced a MSTN KO lamb using CRISPR/Cas9, which suggested that a high level of targeted gene modification could be achieved in goat using CRISPR/Cas9. Taken together, our study indicates that although TALEN enables a variety of genome modifications and may have some advantages over CRISPR/Cas9, the latter provides a significant advantage by permitting precise and efficient gene editing. Thus, CRISPR/Cas9 has more potential to become a robust gene-engineering tool for application in the breeding of farm animals. •We successfully generated an MSTN KO lamb using CRISPR/Cas9.•We compared utility of CRISPR/Cas9 and TALEN for generating Alpas cashmere goats with myostatin (MSTN) gene knockout (KO).•Compared with CRISPR/Cas9 embryos, TALEN MSTN KO embryos reached 8-cell stage at significantly higher cleavage rate.•Compared with TALEN, CRISPR/Cas9 showed greater gene editing efficiency, but with higher number of off-target effects.•CRISPR/Cas9 has high potential to become a