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CRISPR-Cas9 gene editing is dependent on a programmable single guide RNA (sgRNA) that directs Cas9 endonuclease activity. This RNA is often generated by enzymatic reactions, however the process becomes time-consuming as the number of sgRNAs increases and does not allow the incorporation of chemical modifications that can improve or expand the functionality of CRISPR. Solid-phase RNA synthesis can overcome these issues, but highly pure full-length sgRNA remains at the limits of current synthetic methods. Here, we demonstrate a "split-and-click" approach that separates the sgRNA into its two smaller components - a DNA-targeting ~20-mer RNA and a constant Cas9-binding 79-mer RNA - and chemically ligates them together to generate a biologically active sgRNA. The benefits of our approach lie in the stringent purification of the DNA-targeting 20-mer, the reduced synthesis of the constant 79-mer each time a new sgRNA is required, and the rapid access it provides to custom libraries of sgRNAs.

Original publication

DOI

10.1007/978-1-0716-0687-2_5

Type

Journal article

Journal

Methods in molecular biology (Clifton, N.J.)

Publication Date

01/01/2021

Volume

2162

Pages

61 - 78