Recently, T7 Endonuclease I (T7E1) cleavage assay has been widely employed as an efficient approach for detecting mutations from CRISPR/Cas9 targeted samples. This enzyme is sufficient to detect single- and multiple-base mismatches from various heteroduplex DNA samples. However, T7E1 is quite expensive for researchers to use it only for screening mutations, especially in the condition of a large number of test samples. Regarding the production of this enzyme, to data, only the E. coli system has been reported and the highly overexpressed T7E1 seems toxic to the E. coli host cells. Thus, in this study, we tested whether the silkworm-baculovirus expression vector system (BEVS) is suitable to produce recombinant T7 Endonuclease I (rT7E1). The rT7E1 with N- or C-tags in cultured silkworm cells and silkworm pupae were successfully expressed. Our results demonstrated that the rT7E1-Ntag was highly expressed in silkworm pupae and we obtained rT7E1 proteins in high purity. Moreover, rT7E1 from silkworm-BEVS sufficiently recognized and cleaved the mismatches of designed and CRISPR/Cas9-mediated DNA substrates, which was equivalent to the commercial rT7E1 of the E. coli system. Taken together, our study would greatly support the genome-editing research by providing a cost-effective and active rT7E1 enzyme.

最近，T7核酸内切酶I（T7E1）裂解分析已被广泛用作从CRISPR / Cas9靶向样品中检测突变的有效方法。这种酶足以检测来自各种异源双链DNA样品的单碱基和多碱基错配。但是，对于研究人员来说，仅将T7E1用于筛选突变是非常昂贵的，特别是在大量测试样品的情况下。关于这种酶的产生，据数据，仅报道了大肠杆菌系统，而高度表达的T7E1似乎对大肠杆菌有毒。宿主细胞。因此，在这项研究中，我们测试了家蚕杆状病毒表达载体系统（BEVS）是否适合生产重组T7核酸内切酶I（rT7E1）。在培养的蚕细胞和蚕p中成功表达了带有N标签或C标签的rT7E1。我们的结果表明，rT7E1-Ntag在家蚕p中高表达，我们获得了高纯度的rT7E1蛋白。此外，来自蚕-BEVS的rT7E1可以充分识别并切割设计的和CRISPR / Cas9介导的DNA底物的错配，这与大肠杆菌系统的商业rT7E1等效。综上所述，我们的研究通过提供一种经济有效的活性rT7E1酶，将大大支持基因组编辑研究。