sgRNAs

sgRNA stands for single-guide RNA, a short RNA molecule used in CRISPR-Cas9 genome editing. It is a chimeric RNA that fuses the targeting information of the CRISPR RNA (crRNA) with the trans-activating CRISPR RNA (tracrRNA) into a single component that binds the Cas9 nuclease and directs it to a complementary DNA sequence adjacent to a protospacer adjacent motif (PAM).

Typically, an sgRNA contains a 20-nucleotide spacer sequence that determines the genomic target, followed by a conserved scaffold that maintains binding to Cas9. The Cas9-sgRNA complex searches for a PAM and then base-pairs the spacer with the target DNA. Cleavage occurs three base pairs upstream of the PAM, generating a double-strand break that is repaired by the cell, often resulting in indels or, with a donor template, precise edits.

SgRNA design emphasizes target specificity and feasibility. Choices include selecting unique spacers, avoiding sequences with predicted off-target matches, and considering GC content and secondary structure. For SpCas9, the most common PAM is NGG, but many Cas9 and Cas12 variants recognize different PAMs. In practice, sgRNAs can be delivered as DNA constructs, in vitro transcribed RNA, or preformed ribonucleoprotein complexes with Cas9.

Applications include gene knockout, knock-in, and gene regulation when used with catalytically inactive Cas9 (dCas9) fused to transcriptional activators or repressors (CRISPRi/CRISPRa). Limitations include off-target cleavage and delivery challenges; strategies to mitigate these issues include using high-fidelity Cas9 variants, truncated sgRNA designs, and chemical modifications to increase stability.

See also: CRISPR-Cas9, PAM, CRISPRi, CRISPRa.