Spacer sequence for targeting gyrA gene in Bacillus subtilis strain 168 using CRISPR-Cas9

Guide RNA provides unique targeting capability to CRISPR-Cas9 genome editing system through complementary base pairing with the DNA strand at specific locus. Thus, design of the guide RNA sequence plays an important role in achieving precise editing at any location in the genome. But, what defines the guide RNA sequence? The chief component of guide RNA is the spacer sequence that lies immediately adjacent to the protospacer adjacent motif (PAM) sequence in DNA. Design of guide RNA would thus comprise the appending of the spacer sequence to a RNA sequence that forms stem-loop structures complementary to structural motifs in Cas9. Using an in-house MATLAB algorithm, a database comprising all the spacer sequence capable of targeting gyrA gene in Bacillus subtilis strain 168 was created. Collectively, the database of spacer sequences in gyrA gene of B. subtilis 168 should find use in genetic engineering or metabolic engineering studies aiming to modulate gyrA expression through targeted knockdown, knockout or CRISPR mediated gene activation.