CRISPR-Cas9 spacer sequence for targeting ldhA gene in Escherichia coli K-12 MG1655

First evolved as a bacterial adaptive immune system, CRISPR-Cas9 has been co-opted to aid genetic engineering of cells. Specifically, CRISPR-Cas9’s endonuclease capability was utilized to generate precise double strand break in DNA, that could be repaired by a repair sequence (e.g., a replacement gene) through homologous recombination. To use CRISPR-Cas9 genome editing capabilities, a guide RNA (gRNA) is needed to guide the Cas9 endonuclease to the specific location in the genome where a change is to be made. Design of the guide RNA sequence requires knowledge of the 20 nucleotide spacer sequence immediately adjacent to the protospacer adjacent motif (PAM) sequence that anchors Cas9 to the DNA strand. Using an in-house MATLAB algorithm that automatically scan the gene of interest for PAM sequence and outputs a list of spacer sequence immediately adjacent to each PAM sequence, a database of CRISPR-Cas9 spacer sequence for the <i>ldhA</i> gene in <i>Escherichia coli</i> K-12 MG1655 was created. Such spacer sequences could be used to target <i>ldhA</i> for genome editing by CRISPR-Cas9. Collectively, using an in-house MATLAB software, a database of spacer sequences that could be used to target <i>ldhA</i> gene of <i>E. coli </i>K-12 MG1655 by CRISPR-Cas9 was created, and could find use in synthetic biology or metabolic engineering studies seeking to either knockdown or knockout the function of <i>ldhA </i>gene that controls the production of lactate during anaerobic metabolism.<br>