Chang Yi, Huang Honghao, Zhao Ruonan, Diao Lu, Xiong Wenguang, Wan Peng
College of Bioengineering, Hunan Polytechnic of Environment and Biology, Hengyang 421005, China.
Guangdong Provincial Key Laboratory of Veterinary Pharmaceutics Development and Safety Evaluation, South China Agricultural University, Guangzhou, 510642, China; National Risk Assessment Laboratory for Antimicrobial Resistance of Animal Original Bacteria, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China.
Poult Sci. 2025 Aug 15;104(11):105696. doi: 10.1016/j.psj.2025.105696.
Riemerella anatipestifer (R. anatipestifer) is an important pathogen responsible for high mortality rates and severe economic losses in the poultry industry. Research on R. anatipestifer is constrained by limited genetic manipulation tools, highlighting the need for an effective genome editing toolkit. The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 system from Streptococcus pyogenes has been widely used as a genome editing tool for various bacteria, but has not been used in R. anatipestifer. In this study, we developed a CRISPR/Cas9-based genome editing system, pCasRA-SacB, specifically for R. anatipestifer. This shuttle vector contains the replication origin from R. anatipestifer plasmid pRA0726 and p15A origin for replication in both R. anatipestifer and Escherichia coli (E. coli). It also includes chloramphenicol and cefoxitin resistance genes, which allow for selection in E. coli and R. anatipestifer. The vector features BsaI and SalI sites, enabling the cloning of single guide RNA (sgRNA) and homologous arms. In addition, it contains the high-expression promoter of B739_0921 gene and the promoter of rpsL gene, facilitating the expression of sgRNA and Cas9 protein. Furthermore, the vector features the sucrose-sensitive gene sacB and the oriT region, which enable plasmid curing and the transfer of plasmid via conjugation. Notably, the pCasRA-SacB system enables rapid, efficient, and scarless genome editing in R. anatipestifer, including gene deletion, insertion, and point mutation in the dprA gene, with editing efficiencies of 54.2 %, 100.0 %, and 50.0 %, respectively. In summary, the pCasRA-SacB system not only expands the genome editing toolbox of R. anatipestifer but also is helpful in fundamental research in R. anatipestifer.
鸭疫里默氏菌（R. anatipestifer）是一种重要的病原体，在家禽业中可导致高死亡率和严重的经济损失。对鸭疫里默氏菌的研究受到有限的基因操作工具的限制，这凸显了开发一种有效的基因组编辑工具包的必要性。化脓性链球菌的成簇规律间隔短回文重复序列（CRISPR）/Cas9系统已被广泛用作各种细菌的基因组编辑工具，但尚未用于鸭疫里默氏菌。在本研究中，我们专门为鸭疫里默氏菌开发了一种基于CRISPR/Cas9的基因组编辑系统pCasRA-SacB。该穿梭载体包含来自鸭疫里默氏菌质粒pRA0726的复制起点和在鸭疫里默氏菌和大肠杆菌（E. coli）中复制的p15A起点。它还包括氯霉素和头孢西丁抗性基因，可用于在大肠杆菌和鸭疫里默氏菌中进行筛选。该载体具有BsaI和SalI位点，能够克隆单向导RNA（sgRNA）和同源臂。此外，它包含B739_0921基因的高表达启动子和rpsL基因的启动子，有利于sgRNA和Cas9蛋白的表达。此外，该载体具有蔗糖敏感基因sacB和oriT区域，能够实现质粒清除以及通过接合转移质粒。值得注意的是，pCasRA-SacB系统能够在鸭疫里默氏菌中实现快速、高效且无痕的基因组编辑，包括dprA基因的基因缺失、插入和点突变，编辑效率分别为54.2%、100.0%和50.0%。总之，pCasRA-SacB系统不仅扩展了鸭疫里默氏菌的基因组编辑工具箱，而且有助于鸭疫里默氏菌的基础研究。
