Part:BBa_K5490019

gRNA FOR CASRX , SPACER 2 (WNV)

gRNAs (guide RNAs) are relatively small RNA molecules that play a crucial role in gene editing and RNA-targeting technologies. They are typically expressed under the control of a promoter, often polymerase III, which is ideal given the high complexity of the gRNA's secondary structure. These gRNAs are composed of two main components:

The scaffold or direct repeat region: This region forms a complex secondary structure after transcription, allowing it to bind effectively to an effector molecule such as CasRx, which acts as an RNA nuclease, specifically targeting single-stranded RNA. There are two types of direct repeat sequences commonly used--one that is 30 nucleotides long and another that is 36 nucleotides long. The 36-nucleotide direct repeat has been shown to have a higher affinity for CasRx, improving the overall efficiency of the RNA-targeting system.

The spacer: This is a 23-nucleotide sequence that is complementary to the target RNA, guiding CasRx to the specific RNA region for cleavage. In this case, the target is a region within the West Nile Virus (WNV) genome. The spacer sequence was selected using an algorithm designed to optimize its binding and cutting efficiency, ensuring that the CasRx-gRNA complex can cleave the target RNA with high specificity and effectiveness.

By integrating both the scaffold and spacer, researchers can achieve precise RNA cleavage. The CasRx effector dimer, guided by the gRNA, binds to the target RNA region, enabling highly efficient RNA degradation. This optimized approach allows for better control and precision in RNA-targeting applications, such as combating viral infections like WNV or studying gene expression pathways.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Sources

Konermann S, Lotfy P, Brideau NJ, Oki J, Shokhirev MN, Hsu PD. Transcriptome Engineering with RNA-Targeting Type VI-D CRISPR Effectors. Cell. 2018 Apr 19;173(3):665-676.e14. doi: 10.1016/j.cell.2018.02.033. Epub 2018 Mar 15. PMID: 29551272; PMCID: PMC5910255.

Vad-Nielsen J, Lin L, Bolund L, Nielsen AL, Luo Y. Golden Gate Assembly of CRISPR gRNA expression array for simultaneously targeting multiple genes. Cell Mol Life Sci. 2016 Nov;73(22):4315-4325. doi: 10.1007/s00018-016-2271-5. Epub 2016 May 13. PMID: 27178736; PMCID: PMC11108369.

Chuang YF, Wang PY, Kumar S, Lama S, Lin FL, Liu GS. Methods for in vitro CRISPR/CasRx-Mediated RNA Editing. Front Cell Dev Biol. 2021 Jun 11;9:667879. doi: 10.3389/fcell.2021.667879. PMID: 34178991; PMCID: PMC8226256.