Part:BBa_K5490021

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]

Usage and Biology

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.

Engineering and Strutural Experiments

Design Text

We designed a composite array consisting of three spacer regions targeting West Nile Virus (WNV) and two direct repeat (DR) sequences; the other two DRs at the front and back will be provided by the vector. All DR sequences are 36 base pairs in length, except for the last one in the insert, which is 30 base pairs. This adjustment was made as a compromise to reduce the complexity of the insert, facilitating its ordering from the supplier.

Additionally, we introduced BbsI enzyme sites at the borders of the insert, with the cleavage sites facing each other. The chosen vector, pSLQ5429_pUC_hU6-crScaffold_EF1a-BFP, serves as the expression backbone for the gRNA array and contains additional DR sequences downstream of the multi-linker, also incorporating BbsI sites. This design introduces an additional maturation step, enhancing the stability of the gRNA array. Moreover, it prevents the last gRNA from forming complexes with RNAs in the absence of CasRx, thereby providing us with more precise control over the system.

Preparation of the Insert

We performed sub-cloning since the array came in a plasmid. First, we conducted a double digestion on the borders of the ordered plasmid containing the array after validating the efficiency of the BbsI enzyme. We then extracted the insert via gel extraction from a dense gel.

Preparation of the Vector

We performed a double digestion on the vector with the BbsI enzyme, followed by a cleanup using a column to prepare the vector for insertion. This step was crucial because it ensured that religation was not possible, even with different sticky ends. Directional cloning was then performed using T4 ligase, mixing the insert and the vector in a 2:1 molar ratio.

Test Text

We performed restriction digestion assays on multiple colonies after extracting plasmids using alkaline lysis. After several attempts, we identified the correct plasmid in one of the colonies, followed by a midi prep to extract sufficient amounts of the plasmid, which was quantified using a NanoDrop.

The digestions where performed with NdeI and PctI , forming two bands . If we have the wanted insert then this 2 bands are 700 and 500 (like the colony highlighted by the blue line) If we don't have the wanted insert then this 2 bands are 490 and 500 each (like the colony highlighted by the yellow line) Agarose Gel 1,3% Marker: 1kb ladder plus by NEB

Learn Text

Through this process, we learned about the limitations of restriction digestion assays for screening colonies and separating them from background colonies. Given that the insert was relatively small, many colonies produced false negative results, highlighting the need for careful assessment during the screening process.