Golden Gate Cloning of Synthetic CRISPR RNA Spacer Sequences

Prokaryotes use CRISPR-Cas systems to interfere with viruses and other mobile genetic elements. CRISPR arrays comprise repeated DNA elements and spacer sequences that can be engineered for custom target sites. These arrays are transcribed into precursor CRISPR RNAs (pre-crRNAs) that undergo maturation steps to form individual CRISPR RNAs (crRNAs). Each crRNA contains a single spacer that identifies the target cleavage site for a large variety of Cas protein effectors. Precise manipulation of spacer sequences within CRISPR arrays is crucial for advancing the functionality of CRISPR-based technologies. Here, we describe a protocol for the design and creation of a minimal, plasmid-based CRISPR array to enable the expression of specific, synthetic crRNAs. Plasmids contain entry spacer sequences with two type IIS restriction sites and Golden Gate cloning enables the efficient exchange of these spacer sequences. Factors that influence the compatibility of the CRISPR arrays with native or recombinant Cas proteins are discussed.