Using CRISPR/Cas9 Technology to Genetically Transform and Edit the rpsL Amino Acid Sequence of E. Coli Bacteria to Allow it to Grow on Streptomycin Media

The CRISPR/Cas9 system is a naturally-derived gene-editing system that originates from bacterial immune systems. Its main components are the CRISPR-associated protein #9 (Cas9) endonuclease enzyme, a single-guide RNA (sgRNA) made up of CRISPR RNA (crRNA) and tracrRNA, and a protospacer adjacent motif (PAM) sequence (Jinek et al., 2012). Cas9 complexes with the sgRNA to create a ribonuclear protein that binds to template DNA complementary to the target DNA sequence and, following PAM recognition, cuts the DNA at the intended target site. The DNA is repaired with respect to the template DNA, which usually contains a purposeful mutation or base change to achieve the expected result—often a genetic change that expresses a different phenotype or changes the function of the gene.

In a three-day lab experiment—including a preparation day before and a results-measuring/clean-up day after—I successfully used the CRISPR/Cas9 gene-editing system to genetically modify the 43rd amino acid, Lysine (K), in the ribosomal subunit protein (rpsL) amino acid sequence of non-pathogenic DH5ɑ Escherichia coli bacteria, and change it to a Threonine (T). This K43T genetic mutation allowed the bacteria to survive on Luria-Bertani (LB) media containing the antibiotic streptomycin, which normally prevents bacteria from growing because it binds to the ribosomes of the bacteria and inhibits its ability to make proteins. However, the genetic mutation induced by CRISPR stops streptomycin from binding to the bacterial ribosomes, thus allowing the bacteria to grow on the antibiotic-containing LB plate.