洋山港宏病毒组分析揭示CRISPR-Cas系统病毒靶标序列的特异性

Yangshan Harbor Virome Analysis Reveals CRISPR Spacer Targeting Specificity

目的 为了探究CRISPR-Cas系统的靶标特点。方法 运用了包括BALST在内的多种生物信息学分析技术和方法对洋山港宏病毒组数据进行了分析。结果 洋山港宏病毒组数据集中共有25 391条双链DNA病毒序列,其中238条序列上的265个开放读码框(ORF)与134条规律间隔短回文重复(CRISPR)间隔序列产生了315个匹配。经注释后获得了128个ORF和135个匹配的功能信息,占比前5位的依次为终止酶(terminase)、衣壳蛋白(capsid protein)、门蛋白(portal protein)、肽酶(peptidase)和DNA甲基化转移酶(DNA methyltransferase)。匹配多在病毒特定功能基因的保守域或关键结构域中。这表明,CRISPR-Cas系统发挥免疫功能时表现出针对病毒特定基因、功能和结构域的靶标特异性。此外,属于Class 1门类下的Type_I型系统的CRISPR-Cas间隔序列匹配数量远高于其他类型系统,占总数的89.0%。结论 本文的研究结果揭示了CRISPR-Cas系统的靶标偏好,增进了对其的认识,为更好地理解病毒-宿主免疫互作机制提供了新的证据和线索。

Objective Since the first discovery that CRISPR-Cas system provides adaptive immunity of prokaryotic hosts to virus and other mobile genetic elements(MGEs). Numerous studies yielded vital insights into the immune mechanisms, and CRISPR-Cas system has been wildly utilized in gene editing and related research efforts. In the three major immune stages—adaptation, expression and maturation, interference spacer sequences play important roles separately. Although PAM(protospacer adjacent motif) determines the identification of targeted genes by CRISPR-Cas system, what drives the selection of specific genes and reservation on the CRISPR array remains uncertain. To explore the targeting characteristics of CRISPR-Cas systems, the virome of Yangshan harbor surface water and the CRISPR-Cas spacers available in public datasets were subjected to analysis.Methods Based on BLAST searching, viral sequence identification, gene function prediction, and gene conserved domain annotation, the final analysis results were obtained. Results As a result, 25 391 doublestranded DNA viral sequences were identified in the virome;265 open reading frames(ORFs) were predicted for 238 sequences, and 134 CRISPR-Cas spacer sequences yielded 315 viral hits. 128 viral ORFs and 135 hits were functionally annotated, and the top 5 hits including terminase, capsid protein, portal protein, peptidase, and DNA methyltransferase. The matching of spacer(host)-protospacer(virus) often occurred in conserved domains or key structural domains of viral functional genes. Meanwhile, the number of CRISPR-Cas spacer sequence matches for Type_I systems under the Class 1 was much higher than that for other types of systems, accounting for 89.0% of the total. The results show that the CRISPR system will specifically identify and act on key functional genes of the virus. Conclusion The results of this study reveal the targeting specificity of the CRISPR-Cas system,showing new insights and providing new evidence for a better understanding of the mechanisms of virus-host immune interactions.