Development of Tools for Genomic-Editing of the P1 Periplasmic Region of YidC: CRISPR/Cas9 Coupled with Lambda Red Recombineering in Escherichia coli MG1655.

SUMMARY YidC is a conserved and essential protein that plays a role in the insertion and folding of Sec-dependent and Sec-independent membrane proteins. It has six transmembrane domains and one large 35 kDa periplasmic domain (P1) positioned between transmembrane domains 1 and 2. Although YidC is found across all domains of life, only P1 seems to be evolutionarily conserved among Gram-negative bacteria, suggesting that the YidC P1 domain serves an important function. Previous studies have attempted to study the P1 domain of YidC by using P1-specific antisense RNA to knock out expression of YidC; however, this did not eliminate leaky expression of YidC nor allow specific study of the P1 domain. To create a clear genetic background for the study of the P1 domain of YidC, this study aimed to couple Lambda Red Recombineering (LRR) with CRISPR/Cas9 to create a scarless genomic deletion of the P1 domain of YidC. Sequential transformation of LRR and CRISPR/Cas9 machinery into Escherichia coli MG1655 resulted in numerous viable colonies. However, further investigation via colony PCR and Sanger sequencing showed inconclusive results as to whether or not a successful P1 deletion in YidC was generated. Furthermore, while Sanger sequencing indicated successful assembly of CRISPR guide RNA, the CRISPR/Cas9 activity of our cloned pCRISPR appears to be ineffective at killing wild type cells, suggesting that CRISPR/Cas9 effectiveness is highly dependent on the target site of the gRNA. This study provides useful insight for futures studies hoping to make genomic deletions in essential genes using the CRISPR/Cas9 system in tandem with Lambda Red Recombineering.