基于CRISPR/Cas9系统筛选猪流行性腹泻病毒复制相关基因及其验证

Screening and validation of porcine epidemic diarrhea virus replication-related genes based on genome-scale CRISPR/Cas9 system

摘要:

【背景】成簇规律间隔的短回文重复序列相关蛋白(clustered regularly interspaced short palindromic repeats/CRISPR-associated protein，CRISPR/Cas9)已被广泛证实是高效、强大的第三代基因编辑工具，在发现功能基因等领域取得了重要进展，但至今尚无利用该方法挖掘猪流行性腹泻病毒(porcine epidemic diarrhea virus，PEDV)宿主基因的报道。【目的】利用CRISPR/Cas9系统在全基因组范围内筛选PEDV复制相关基因，并进行候选基因的初步验证，为培育抗PEDV种猪提供科学参考。【方法】通过CRISPR/Cas9技术构建人肝癌细胞系(Huh-7)全基因组敲除文库，利用PEDV感染Huh-7文库细胞，随后经过高通量测序筛选影响PEDV复制的关键宿主因子，结合基因干扰和检测病毒效价等相关试验对影响PEDV复制的候选基因进行初步验证。【结果】构建了CRISPR/Cas9系统在全基因组范围内筛选PEDV复制相关基因的方法，将富集程度排名靠前的整合素α11(integrin α11，ITGA11)、哺乳动物复制蛋白A2(replication protein A2，RPA2)、驱动蛋白家族成员2A(kinesin family member 2A，KIF2A)、诱导髓系白血病细胞分化蛋白1(induced myeloid leukemia cell differentiation protein 1，MCL1)、多聚ADP核糖化酶1[poly(ADP-ribose)polymerase 1，PARP1]和囊泡单胺转运蛋白(vesicular monoamine transporter，SLC18A1)基因进行了验证；采用siRNA对上述基因分别进行干扰后，结果与对照组相比，干扰ITGA11可显著降低PEDV猪源靶向细胞IPEC-J2中PEDV-NmRNA、蛋白表达水平及子代病毒滴度。【结论】基于CRISPR/Cas9系统的全基因组敲除文库可作为挖掘PEDV复制相关功能基因的有效工具，ITGA11基因可作为一种制备抗PEDV猪种潜在的靶基因。

关键词:成簇规律间隔的短回文重复序列相关蛋白(CRISPR/Cas9);猪流行性腹泻病毒;遗传筛选;整合素α11

Abstract:

[Background] Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas9) has been proven to be an efficient and powerful third-generation gene editing tool and has achieved great progress in the discovery of functional genes. However, few studies have reported about using this method to screen the host genes associated with porcine epidemic diarrhea virus (PEDV). [Objective] To screen out the genes related to PEDV replication in the whole genome by using the CRISPR/Cas9 system, and to perform preliminary verification of candidate genes, so as to provide scientific reference for breeding PEDV-resistant pigs. [Methods] A genome-wide knockout library of human hepatoma cell line (Huh-7) was constructed via CRISPR/Cas9 technology, and the Huh-7 library cells were infected with PEDV. Then, the key host factors affecting PEDV replication were screened by high-throughput sequencing. The interference and detection experiments of virus replication were performed to preliminarily verify the candidate genes that affected PEDV replication. [Results] A CRISPR/Cas9 system was successfully constructed to screen the genes related to PEDV replication in the whole genome. The genes with top enrichment degree, including those encoding integrin α11 (ITGA11), replication protein A2 (RPA2), kinesin family member 2A (KIF2A), induced myeloid leukemia cell differentiation protein 1 (MCL1), poly (ADP-ribose) polymerase 1 (PARP1), and solute carrier family 18 member A1 (vesicular monoamine transporter, SLC18A1), were verified. Compared with the control group, the interference of ITGA11 via siRNA significantly down-regulated the mRNA and protein levels of PEDV-N and reduced the virus titer in the IPEC-J2 cells infected with PEDV. [Conclusion] The genome-wide knockout library based on the CRISPR/Cas9 system can be used as an effective tool for screening PEDV replication-related functional genes. ITGA11 may be a potential target gene for the breeding of PEDV-resistant pigs.

Key words:clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas9);porcine epidemic diarrhea virus (PEDV);genetic screening;integrin α11 (ITGA11)