Assembling Method Of RNA(sgRNA) For Streptococcus Thermophilus CRISPR Mediated Multiplex Targeting

CRISPR/Cas has been emerged as a powerful and versatile platform for biotechnological, biomedical and transgenic researches. In addition to the advantages for scalable, affordable and easy to engineer, the clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein(Cas) technology promises a superiority for multiplex targeting. However, current popular usages for multiplex targeting all preferred to involve multiple gRNA expressing cassettes, which are very laborious and inconvenient to clone and assemble. We developed a simple method to artificially assemble the Streptococcus thermophilus CRISPR array based on compatible restriction enzyme strategy, which will facilitate the multiplex targeting usage for the CRISPR/Cas technology, and furthermore provide a potential tool for preparing efficient sequence-specific antimicrobials. First, the Streptococcus thermophilus CRISPR3/Cas locus was cloned. Second, different CRISPR arrays were assembled with different crRNA spacers. Transformation assays using different Escherichia colistrains demonstrated efficient plasmid DNA targeting, and we achieved targeting efficiency up to 95% with an assembled CRISPR array with three crRNA spacers.