Dong Z, Hu Z, Qin Q, Dong F, Huang L, Long J, Chen P, Lu C, Pan M
State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, 400716, China.
Key Laboratory of Sericultural Biology and Genetic Breeding, Ministry of Agriculture, Southwest University, Chongqing, 400716, China.
Insect Mol Biol. 2019 Feb;28(1):112-122. doi: 10.1111/imb.12529. Epub 2018 Oct 8.
The CRISPR/Cas9 system is a powerful tool for the treatment of infectious diseases. In our previous study, we knocked out the Bombyx mori nucleopolyhedrovirus (BmNPV) key genes and BmNPV-dependent host factor to generate transgenic antiviral strains. To further expand the range of target genes for BmNPV and more effectively prevent and control pathogenic infections, we performed gene editing and antiviral analysis by constructing a target-directed baculovirus early transcriptional activator immediate early-0 (ie-0) and 2 (ie-2) transgenic silkworm line. We hybridized it with Cas9 transgenic line to produce a double-positive transgenic Cas9(+)/sgIE0-sgIE2(+) line that could activate the CRISPR gene editing system. We first demonstrated that the system is capable of efficiently editing target genes and resulting in fragment deletions in the BmNPV genome. Survival rate of the transgenic Cas9(+)/sgIE0-sgIE2(+) line reached 65% after inoculation with 1 × 10 occlusion bodies/larva. Molecular analysis showed that BmNPV DNA replication and viral gene expression level in the transgenic Cas9(+)/sgIE0-sgIE2(+) line were significantly inhibited compared with the control Cas9(-)/sgIE0-sgIE2(-) line. These results indicated that IE-0 and IE-2, as baculovirus early transcriptional activators, can be used as target sites for gene therapy and that multigene editing could expand the range of target sites for research to create silkworm resistance breeds.
CRISPR/Cas9系统是治疗传染病的有力工具。在我们之前的研究中，我们敲除了家蚕核型多角体病毒（BmNPV）的关键基因和BmNPV依赖的宿主因子，以产生转基因抗病毒品系。为了进一步扩大BmNPV的靶基因范围，更有效地预防和控制病原体感染，我们通过构建靶向杆状病毒早期转录激活因子立即早期蛋白-0（ie-0）和立即早期蛋白-2（ie-2）转基因家蚕品系进行了基因编辑和抗病毒分析。我们将其与Cas9转基因品系杂交，产生了一个双阳性转基因Cas9(+)/sgIE0-sgIE2(+)品系，该品系可以激活CRISPR基因编辑系统。我们首先证明该系统能够高效编辑靶基因，并导致BmNPV基因组中的片段缺失。用1×10个包涵体/幼虫接种后，转基因Cas9(+)/sgIE0-sgIE2(+)品系的存活率达到65%。分子分析表明，与对照Cas9(-)/sgIE0-sgIE2(-)品系相比，转基因Cas9(+)/sgIE0-sgIE2(+)品系中的BmNPV DNA复制和病毒基因表达水平受到显著抑制。这些结果表明，作为杆状病毒早期转录激活因子的IE-0和IE-2可作为基因治疗的靶点，多基因编辑可以扩大创建抗蚕品种的研究靶点范围。
