Difference between revisions of "Part:BBa K5490021"

 
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<partinfo>BBa_K5490021 parameters</partinfo>
 
<partinfo>BBa_K5490021 parameters</partinfo>
 
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gRNA arrays offer a powerful multi-targeting approach for RNA manipulation, enabling the simultaneous targeting of multiple RNA sequences. In this particular setup, the gRNA array consists of three individual gRNAs, each subdivided into two components: the scaffold (which forms the structure required to bind CasRx) and the spacer (which is complementary to the target RNA sequence). These spacers are designed to target three distinct sites within the West Nile Virus (WNV) genome, selected for their high specificity using an algorithmic approach.
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Once expressed, the gRNA array interacts with CasRx, which facilitates the processing of the array into three mature individual gRNAs. This maturation process generates three distinct CasRx-gRNA complexes, each targeting a different site in the WNV genome. By modifying the spacer sequences, researchers can customize the array to target different RNA sequences of interest, making the system adaptable for a variety of RNA-targeting applications.
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Compared to individual gRNAs, the array offers several advantages:
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Multi-targeting capability: The array allows for the simultaneous targeting of multiple RNA sequences, increasing the efficiency of RNA silencing or viral inhibition.
 +
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Reduced off-target effects: The maturation step required for the gRNA array to function provides an additional layer of control, as it is dependent on the concentration of CasRx. This step ensures that the array is processed into functional gRNAs only when CasRx is present at sufficient levels, reducing the likelihood of off-target activity.
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Sources
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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.
 +
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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.
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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.

Revision as of 17:07, 27 September 2024


gRNA ARRAY FOR CASRX , SPACER 1,2,3 (WNV)

It is a nucleic acid sequence with three complementary regions targeting the WNV genome. After expression, the RNA interacts with CasRx protein, undergoes maturation, and targets three separate regions of the WNV genome. Researchers can modify these three 23-nt regions to enable multi-target cleavage in any RNA of their choice

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]


gRNA arrays offer a powerful multi-targeting approach for RNA manipulation, enabling the simultaneous targeting of multiple RNA sequences. In this particular setup, the gRNA array consists of three individual gRNAs, each subdivided into two components: the scaffold (which forms the structure required to bind CasRx) and the spacer (which is complementary to the target RNA sequence). These spacers are designed to target three distinct sites within the West Nile Virus (WNV) genome, selected for their high specificity using an algorithmic approach.

Once expressed, the gRNA array interacts with CasRx, which facilitates the processing of the array into three mature individual gRNAs. This maturation process generates three distinct CasRx-gRNA complexes, each targeting a different site in the WNV genome. By modifying the spacer sequences, researchers can customize the array to target different RNA sequences of interest, making the system adaptable for a variety of RNA-targeting applications.

Compared to individual gRNAs, the array offers several advantages:

Multi-targeting capability: The array allows for the simultaneous targeting of multiple RNA sequences, increasing the efficiency of RNA silencing or viral inhibition.

Reduced off-target effects: The maturation step required for the gRNA array to function provides an additional layer of control, as it is dependent on the concentration of CasRx. This step ensures that the array is processed into functional gRNAs only when CasRx is present at sufficient levels, reducing the likelihood of off-target activity.

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.