Oxford Nanopore long-read genome sequencing of mutants in Streptococcus suis strain P1/7 constructed using pSStarget, a novel plasmid-based CRISPR/Cas9 genome editing system

<strong>Description:</strong>This dataset contains ONT sequencing reads of the wild type strain <em>S. suis</em> P1/7 and four derived mutant strains constructed using a novel CRISPR-Cas9 genome editing system for S. suis. Our data shows that our laboratory stock of strain P1/7 has 2 SNPs and a 5 bp deletion relative to the reference sequence (AM946016.1) published in GenBank. We confirm that the precise deletion of the <em>cpsEF </em>and s<em>ly</em> genes in the respective mutants as well as in the double knockout mutant. Moreover, we confirm the precise introduction of targeted mutations resulting in a single amino acid change in the Enolase gene product. No other mutations or structural variations have been detected. <strong>This dataset contains the following 5 files:</strong>WT_raw_reads.fastq.gz dCps_raw_reads.fastq.gz dSly_raw_reads.fastq.gz dCps_dSly_raw_reads.fastq.gz enoK261A_raw_reads.fastq.gz <strong>Explanation of variables:</strong>All files contain raw ONT reads in the FASTQ (.fq) format in zipped (.gz) format.WT refers to the wild type strain Streptococcus suis P1/7. The prefix dCps and dSly refers to a knockout strain with the <em>cpsE/cpsF</em> and <em>sly</em> genes deleted, respectively. The double knockout mutant, in which both loci were deleted, is denoted with the prefix dCps_dSly. Finally, prefix enoK261A denotes the strain with a single amino acid mutation in the essential <em>eno</em> gene.<br><strong>Materials and methods:</strong>All strains were grown in Todd-Hewitt broth (Oxoid) supplemented with 0.2% Bacto™ yeast extract (BD Biosciences) (THY) without agitation. Overnight cultures (10ml) were harvested by centrifugation at 4500x for 10min and the pellets were transferred to bead beating tubes tube containing 0.1mm silica beads (Lysing matrix B, MP Biomedicals) and lysed by bead beating for 40 seconds at 4.0 m/s using a FastPrep-24 5G (MP Biomedicals). The lysates were cleared by centrifugation at 16.000x for 10 minutes and the genomic DNA (gDNA) has been isolated using the PowerSoil DNA Isolation Kit (Qiagen) according to manufacturer’s instructions. The concentration, purity and integrity of the extracted gDNA were determined using the Qubit BR DNA assay, DeNovix spectrophotometer and gel electrophoresis, respectively. The concentrations were normalized to 50ng/μl and sent to Plasmidsaurus (https://www.plasmidsaurus.com/) for further processing. Briefly, genomic DNA was minimally fragmented, amplification-free libraries were constructed from the fragmented DNA using the v14 library prep chemistry and the libraries were sequenced using R10.4.1 flow cells. The raw reads have been delivered in FASTQ format.<br><strong>License</strong>This dataset is published under the CC BY-SA (Attribution ShareAlike) license.<br>

**数据集描述：** 本数据集包含野生型猪链球菌（*Streptococcus suis*, S. suis）菌株P1/7以及四种基于新型CRISPR-Cas9基因组编辑系统构建的该菌株衍生突变株的牛津纳米孔测序技术（Oxford Nanopore Technologies, ONT）测序读段。本研究数据显示，本实验室保存的P1/7菌株与GenBank中发布的参考序列（AM946016.1）相比，存在2个单核苷酸多态性（Single Nucleotide Polymorphism, SNP）位点以及一段5碱基缺失。我们确认，各单基因敲除突变株以及双基因敲除突变株中均精准缺失了*cpsEF*与*sly*基因；同时确认，烯醇化酶（Enolase）基因产物中成功引入了靶向突变，实现了单个氨基酸的替换。未检测到其他突变或结构变异。

**本数据集包含以下5个文件：**
WT_raw_reads.fastq.gz、dCps_raw_reads.fastq.gz、dSly_raw_reads.fastq.gz、dCps_dSly_raw_reads.fastq.gz、enoK261A_raw_reads.fastq.gz

**变量说明：**
所有文件均为压缩（.gz）格式的FASTQ（.fq）格式原始ONT测序读段。WT代表野生型猪链球菌菌株*Streptococcus suis* P1/7；前缀dCps与dSly分别代表*cpsE/cpsF*与*sly*基因敲除菌株；同时缺失两个靶位点的双基因敲除突变株以dCps_dSly作为前缀；最后，前缀enoK261A代表在必需基因*eno*（烯醇化酶基因）中携带单个氨基酸突变的菌株。

**材料与方法：**
所有菌株均在添加了0.2% Bacto™酵母提取物（BD生物科学公司，BD Biosciences）的托德-希维特肉汤（Oxoid品牌，THY）中静置培养。过夜培养物（10ml）经4500×g离心10分钟收集菌体，将沉淀转移至装有0.1mm硅胶微珠（裂解基质B，MP生物医学公司，MP Biomedicals）的珠磨管中，使用FastPrep-24 5G珠磨破碎仪（MP生物医学公司）以4.0 m/s的速率珠磨破碎40秒完成裂解。裂解液经16000×g离心10分钟澄清后，使用PowerSoil DNA提取试剂盒（凯杰公司，Qiagen）按照制造商说明书提取基因组DNA（gDNA）。使用Qubit BR DNA定量试剂盒、DeNovix分光光度计与凝胶电泳分别测定提取的gDNA的浓度、纯度与完整性。将DNA浓度归一化至50ng/μl后，送至Plasmidsaurus公司（https://www.plasmidsaurus.com/）进行后续处理。简要流程如下：将基因组DNA进行轻度片段化，采用v14文库制备试剂构建无需扩增的片段化DNA文库，使用R10.4.1测序流通池完成文库测序。原始读段以FASTQ格式交付。

**许可协议：**
本数据集采用CC BY-SA（署名-相同方式共享，Attribution ShareAlike）许可协议发布。