Development and implementation of a Type I-C CRISPR-based Programmable Repression System for Neisseria gonorrhoeae

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) are prokaryotic adaptive immune systems regularly utilized as DNA editing tools. While Neisseria gonorrhoeae does not have an endogenous CRISPR, the commensal species Neisseria lactamica encodes a functional Type I-C CRISPR-Cas system. We have established an IPTG-inducible, CRISPR-interference (CRISPRi) platform based on the N. lactamica Type I-C CRISPR missing the Cas3 nuclease to allow locus-specific transcriptional repression. As proof of principle, we targeted a non-phase-variable version of the opaD gene. We show that CRISPRi can downregulate opaD gene and protein expression, resulting in bacterial inability to stimulate neutrophil oxidative responses and to bind to an N-terminal fragment of CEACAM1. Importantly, we used CRISPRi to effectively knockdown all the transcripts of all eleven opa genes using a five-spacer CRISPR array, allowing control of the entire phase-variable opa family in strain FA1090. We also report that repression is reversible following IPTG-removal. Finally, we showed that the Type I-C CRISPRi system can conditionally reduce the expression of two essential genes. This CRISPRi system will allow the interrogation of every Gc gene, essential and non-essential, to study physiology and pathogenesis, and aid in antimicrobial development.

成簇规律间隔短回文重复序列（CRISPR，Clustered Regularly Interspaced Short Palindromic Repeats）是原核生物适应性免疫系统，常被用作DNA编辑工具。尽管淋病奈瑟菌（Neisseria gonorrhoeae）不具有内源性CRISPR系统，但共生菌种乳糖奈瑟菌（Neisseria lactamica）却编码有功能的I-C型CRISPR-Cas系统。本研究基于缺失Cas3核酸酶的乳糖奈瑟菌I-C型CRISPR系统，构建了异丙基-β-D-硫代半乳糖苷（IPTG）诱导型CRISPR干扰（CRISPRi）平台，可实现位点特异性的转录抑制。作为原理验证，我们靶向了opaD基因的非相变异版本。实验结果表明，CRISPRi可下调opaD基因及其编码蛋白的表达，使细菌无法刺激中性粒细胞氧化应答，也无法结合CEACAM1的N端片段。尤为重要的是，我们利用五间隔区CRISPR阵列，借助CRISPRi技术成功敲低了11个opa基因的全部转录本，从而实现了对FA1090菌株中整个相变异opa基因家族的调控。此外，我们还发现去除IPTG后，基因抑制效应可逆转。最后，实验证实该I-C型CRISPRi系统能够条件性降低两个必需基因的表达。该CRISPRi系统可用于探究每一个淋病奈瑟菌（Gc）基因（包括必需与非必需基因）的功能，以研究其生理特性与致病机制，并助力抗菌药物的开发。