Single-guide RNA (sgRNA) is the critical RNA component of the cas9 CRISPR-Cas9 genome-editing system, where it base-pairs with target nucleic acids and directs Cas9 to specific genomic loci for precise DNA modification. It is a gene-editing RNA analyte used across genome editing, base editing, and analytical quality-control workflows, including nanopore direct RNA sequencing and impurity profiling. Recent work showed full-length sequencing of 100-nucleotide sgRNA molecules by adding a 5′ RNA oligo adapter, and sgRNA identity could be verified by nanopore direct RNA sequencing. In delivery applications, sgRNA has been co-delivered with Cas9 mRNA by ah lnp for pancreatic genome editing, and with base editor mRNA by electroporation in an optimized editing system. sgRNA has also been multiplexed, with four sgRNAs assembled tandemly into one Cas9 plasmid to target donor or acceptor DNA and distinguish HSA21 copies in a Down syndrome study.
Genome editing and delivery
- sgRNA guides cas9 to specific genomic loci for precise DNA modification, defining its core role in CRISPR-Cas9 editing. (PMID:41952066)
- Guide RNA delivered with Cas9 mRNA by ah lnp enabled precise and efficient pancreatic genome editing. (PMID:41741655)
- Single guide RNA was delivered by electroporation together with base editor mRNA in an optimized editing system. (PMID:42018671)
- In a CRISPR/Cas9 strategy for Down syndrome, four sgRNAs were assembled tandemly into one Cas9 plasmid to target donor or acceptor DNA and distinguish HSA21 copies. (PMID:41973928)
Analytical characterization and quality control
- Nanopore direct RNA sequencing was used to sequence and identity-verify guide RNA molecules, including full-length 100-nucleotide sgRNA molecules. (PMID:41787951)
- Full-length sequencing of 100-nucleotide sgRNA molecules was demonstrated by incorporating a 5′ RNA oligo adapter. (PMID:41787951)
- sgRNA impurity fractions were profiled using IP-RPLC, SEC, native mass spectrometry, and nanopore direct RNA sequencing. (PMID:41952066)
- Functional assessment of sgRNA impurities was performed with the Cas-CLOVER system to evaluate their impact on performance. (PMID:41952066)
Genome editing applications
- sgRNA is used with cas9 to target desired nucleic acids in CRISPR-Cas genome editing. (PMID:40833724)
- In a 2025 Molecular Biotechnology review, sgRNA was highlighted as part of CRISPR-Cas systems being advanced toward eradication of human microbial pathogens. (PMID:40833724)
- sgRNA-optimized base editing was used in a 2026 Science Translational Medicine study aimed at correcting the CFTR 1717-1G>A mutation. (PMID:42018671)
- Multiplex sgRNA design was applied to selectively target donor or acceptor DNA in a chromosomal silencing approach for Down syndrome. (PMID:41973928)