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Scaffold or direct repeat region (DR 30)

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.

By integrating both the scaffold and spacer, researchers can achieve precise RNA cleavage

Sequence and Features

Source

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.