Greetings Everyone,

I have been reviewing numerous articles and protocols related to Golden Gate assembly for cloning CRISPR arrays for Cas13. Despite my research, there remains one aspect that I find puzzling. Initially, the guide RNA is cloned into a single plasmid and then cut with an overhang, which should complement another cutting guide from a different plasmid. Together, these pieces fit like a puzzle and form the CRISPR array.

My query is as follows: Why is it necessary to clone the single guide into a plasmid, cut it, and then clone it into the final plasmid? Would it not be more efficient to order ssDNA, anneal them, and retain the overhang we desire, rather than cloning into one plasmid and subsequently into another?

Has anyone encountered a similar issue or have insights to share? I would greatly appreciate any advice or suggestions concerning this matter. Looking forward to your responses and assistance.

Sincerely,

Anan