Part:BBa_K5490003
23-nt sequence binds CasRx to cleave WNV genome; modifiable target 3
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
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. This is the third spacer sequence.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
The gRNAs are complex in their design, particularly because they are part of a composite system. Each gRNA is expressed as a polycistronic RNA, which, upon expression, interacts with the CasRx protein. This interaction leads to the maturation of the gRNA and its subsequent cleavage into three distinct gRNAs, each targeting different regions of the virus.
Given this complexity, it is crucial to validate the functionality of each individual gRNA before assembling them into the composite array. The gRNA array consists of two main components: Direct Repeats (DRs)
Spacer Sequences: These sequences are complementary to specific regions of the virus and guide the endonuclease activity of CasRx to its target.
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.
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