Spacer sequences for targeting lactate dehydrogenase (ldhA) in Escherichia coli K-12 through CRISPR-Cas9 genome editing

Cluster regularly interspersed short palindromic repeats (CRISPR)-Cas9 is the dominant genome editing technique in contemporary biological research. The approach requires a guide RNA encoding a spacer sequence to target a specific location in the genome for editing; thereby, necessitating design for the spacer sequence. Typically, the spacer sequence is immediately adjacent to the protospacer adjacent motif (PAM). This work illustrates the approach by using an in-house MATLAB software to design the spacer sequence that would target the lactate dehydrogenase gene (ldhA) in Escherichia coli K-12 for editing. Specifically, the 20 nucleotide spacer sequences could be either on the sense and antisense DNA strands for the gene. Overall, this dataset should find use in biotechnology or metabolic engineering applications where the objective is to delete the ldhA gene to help redirect metabolic flux away from mixed acid fermentation during anaerobic growth of E. coli K-12 in glucose-based medium.

规律间隔成簇短回文重复序列（Cluster regularly interspersed short palindromic repeats, CRISPR）-Cas9是当代生物研究领域主流的基因组编辑技术。该技术需借助一段编码间隔序列（spacer sequence）的向导RNA（guide RNA）靶向基因组中的特定位点以完成编辑，因此需要对间隔序列进行设计。通常，间隔序列紧邻原间隔序列毗邻基序（protospacer adjacent motif, PAM）。本研究采用自研MATLAB软件，设计靶向大肠杆菌K-12（Escherichia coli K-12）乳酸脱氢酶基因（ldhA）的间隔序列，以此演示该编辑方法的实现流程。具体而言，针对该基因的20个核苷酸长度的间隔序列，可分别位于DNA的有义链与反义链。总体而言，本数据集可应用于生物技术或代谢工程相关场景，其目标为敲除大肠杆菌K-12的ldhA基因，以在葡萄糖基培养基中进行厌氧培养时，帮助将代谢流从混合酸发酵途径进行重定向。