The programmable RNA component that directs CRISPR-Cas9 and related Cas nucleases to a specific genomic DNA or mRNA target via Watson–Crick base-pairing. Following Elitza Deltcheva and Emmanuelle Charpentier's 2011 discovery that trans-activating CRISPR RNA (tracrRNA) base-pairs with CRISPR RNA (crRNA) for maturation, Martin Jinek, Jennifer Doudna, Emmanuelle Charpentier, and colleagues engineered the two RNAs in 2012 into a single chimeric transcript — the single guide RNA (sgRNA) — reducing a three-component bacterial defense system into a two-component genome-editing tool.
Class
Programmable targeting and nuclease-scaffolding non-coding RNA (crRNA:tracrRNA or chimeric sgRNA)
Typical Length
~100 nucleotides for standard SpCas9 sgRNA (20-nt 5' target spacer + ~80-nt 3' Cas9-binding scaffold)
Switches Cas nuclease into active conformation and hybridizes 20-nt spacer to target nucleic acid
Clinical / Biotech Use
Genome-wide CRISPR knockout screens, chemically modified clinical sgRNAs, and prime editing pegRNAs
Structure & Biogenesis
In native Type II bacterial immunity, a guide RNA is a dual-RNA hybrid composed of a 42-nucleotide crRNA (carrying a 20-nucleotide variable 5' spacer derived from phage DNA and a 22-nucleotide repeat region) base-paired to an ~89-nucleotide tracrRNA processed by host RNase III (hydrolase).
In engineered SpCas9 sgRNAs, a four-nucleotide GAAA tetraloop covalently links the 3' end of the crRNA repeat to the 5' end of the tracrRNA anti-repeat, creating a single ~100-nucleotide molecule with a 5' variable 20-nt spacer followed by a lower stem, bulge, upper stem, nexus, and two 3' terminal hairpins.
Unlike Cas9, Type V Cas12a (Cpf1) enzymes require only a single short ~42-nt crRNA without a tracrRNA and process their own crRNA arrays using an intrinsic ribonuclease domain.
Mechanism of Action
Binding of the sgRNA 3' scaffold — specifically the repeat:anti-repeat duplex and the conserved nexus stem-loop — induces a large conformational rearrangement in CRISPR-Cas9 that pre-organizes nucleotides 10 to 20 of the spacer (the PAM-proximal "seed" region) into an A-form helical conformation exposed to solvent. Once Cas9 locates a 5'-NGG-3' PAM on double-stranded DNA in the nucleus, the guide RNA seed nucleates directional base-pairing with the complementary DNA target strand, displacing the non-target strand into an R-loop; mismatches within the 8-to-12-nt seed abolish binding, whereas PAM-distal mismatches at positions 18 to 20 are more tolerated and govern off-target cleavage profiles.
Biological & Regulatory Roles
Before CRISPR-Cas9, the term guide RNA (gRNA) was coined in 1990 for short mitochondrial transcripts in kinetoplastid protozoa (such as Trypanosoma brucei) that direct post-transcriptional uridine insertion and deletion editing of mitochondrial mRNAs.
In eukaryotic cells, eukaryotic small nucleolar RNAs (snoRNAs), siRNAs, and miRNAs follow the same fundamental design principle: an RNA guide module conferring sequence specificity to a protein effector complex.
Interactions & Complexes
Scaffold hairpins of an sgRNA can be extended with bacteriophage MS2 or PP7 RNA aptamer loops to recruit transcriptional activators, epigenetic Histone H3 modifiers, or fluorescent proteins to specific genomic loci.
Prime editing guide RNAs (pegRNAs) append a 3' primer-binding site and a reverse-transcription template to the sgRNA, enabling templated insertions, deletions, and all 12 point mutations without double-strand breaks or donor DNA.
Therapeutic & Biotechnology Applications
For plasmid or lentiviral pooled CRISPR screens, sgRNAs are transcribed inside human cells from RNA polymerase III promoters (typically the human U6 snRNA promoter).
For clinical stem-cell editing and lipid nanoparticle delivery to the liver, solid-phase synthesized 100-nt sgRNAs incorporate 2'-O-methyl-3'-phosphorothioate modifications on the terminal three nucleotides at both the 5' and 3' ends to prevent degradation by intracellular ribonucleases.