Part:BBa_K5490000

23-nt sequence binds CasRx to cleave WNV genome modifiable target 1.

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 first spacer sequence.

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]

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): These sequences produce secondary structures that are essential for the CasRx protein to bind, initiating the maturation process of the gRNAs. Spacer Sequences: These sequences are complementary to specific regions of the virus and guide the endonuclease activity of CasRx to its target. By validating each gRNA separately, we ensure that each component of the array functions correctly before combining them into the final composite part. This step is essential for achieving precise targeting and effective cleavage of the viral genome. First, for the individual gRNAs, we ordered three separate inserts, each consisting of a direct repeat (DR) sequence upstream and a specific spacer sequence downstream. These inserts were designed with recognition sites for the Type IIS restriction enzyme BbsI, positioned at both the 5' and 3' ends. This design ensures that after cleavage, the recognition sites are removed, leaving only the desired gRNA sequence. The BbsI recognition sites were placed facing each other to facilitate this separation.

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.