CRISPR/Cas9-mediated disruption of the immediate early-0 and 2 as a therapeutic approach to Bombyx mori nucleopolyhedrovirus in transgenic silkworm
CRISPR/Cas9-mediated disruption of the immediate early-0 and 2 as a therapeutic approach to Bombyx mori nucleopolyhedrovirus in transgenic silkworm
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CRISPR/Cas9介导的立即早期0和2破坏作为转基因家蚕核多角体病毒的治疗方法
DOI：
10.1111/imb.12529
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发表时间：
2019-02-01
影响因子：
2.6
通讯作者：
Pan, M.
中科院分区：
文献类型：
--
作者：
Dong, Z.;Hu, Z.;Pan, M.
CRISPR/Cas9系统是治疗感染性疾病的有力工具。在我们前期的研究中，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，获得了转基因抗病毒株。为了进一步扩大家蚕核型多角体病毒的靶基因范围，更有效地防控病原性感染，我们构建了靶向杆状病毒早期转录激活因子立即早期0（ie-0）和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转基因株系杂交，产生了能够激活CRISPR基因编辑系统的双阳性转基因Cas9（+）/sgIE 0-sgIE 2（+）株系。我们首先证明该系统能够有效编辑靶基因并导致BmNPV基因组中的片段缺失。转基因Cas9（+）/sgIE 0-sgIE 2（+）系在以1 × 10（6）个包合体/幼虫接种后存活率达到65%。分子生物学分析表明，与对照Cas9（-）/sgIE 0-sgIE 2（-）株系相比，转基因Cas9（+）/sgIE 0-sgIE 2（+）株系中BmNPV DNA的复制和病毒基因的表达水平受到显著抑制。这些结果表明，IE-0和IE-2作为杆状病毒的早期转录激活因子，可以作为基因治疗的靶位点，多基因编辑可以扩大研究的靶位点范围，以创建家蚕抗性品种。
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 x 10(6) 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.