RNA

Part:BBa_K5036011

Designed by: Emad hamdy Matter   Group: iGEM24_AFCM-Egypt   (2024-09-12)
Revision as of 17:33, 30 September 2024 by Em100 (Talk | contribs) (→‎Literature Characterization)


Nanog gRNA2 of dCas9

Part Description

It is guide RNA which is a crucial component of the CRISPR-Cas9 gene editing system as it act as A guide the dCas9 to its target. It consist of two main parts: first Spacer Sequence: which is a short, user-defined sequence that complements the target DNA sequence you want to edit. Second Scaffold Sequence which is pre-designed RNA structure allows gRNA to bind to the Cas9 protein and facilitate its interaction with the target DNA

Usage

after the fusion of the two parts of dCas9, it will guide the (dCas9, VP64, GAL4, UAS Trans CMV enhancer) complex to the target DNA of Nanog gene which is located upstream to of YAP-1 so that they can initiate transcription and boost YAP-1 production.

this figure illustrates the structure of Nanog gRNA where it direct (dCas9, VP64, GAL4, UAS Trans CMV enhancer) complex to the target DNA of Nanog gene for boosing YAP-1 production .

Literature Characterization

To evaluate dCas9-VPR's ability to reactivate silenced pluripotency genes, the NANOG gene was targeted in human cells. Two guide RNAs (gRNAs) were designed to bind upstream of the NANOG transcription start site. A GFP-2A-Puromycin cassette was included to track transfection efficiency and select for successfully modified cells. We then co-transfected 293FT cells with dCas9-VPR and gNANOG plasmids. Control experiments were conducted using dCas9-VPR and a TetO-targeting gRNA.

This figure shows The NANOG guide RNA (gRNA) binding sites were positioned 254 and 144 base pairs upstream of the NANOG transcription start site (TSS). The protospacer-adjacent motif (PAM) sequences are highlighted in red, and the exons are indicated by black boxes .


This figure shows thatThe upper images show fluorescent microscopy results of transfected cells. Cells transfected with the gNANOG plasmid displayed intense green fluorescence. The lower graph presents flow cytometry data on the percentage of GFP-positive cells in each group .

Reference

Jianying Guo, Dacheng Ma, Rujin Huang, Jia Ming, Min Ye, Kehkooi Kee, Zhen Xie, Jie Na, An inducible CRISPR-ON system for controllable gene activation in human pluripotent stem cells, Protein & Cell,Volume 8, Issue 5, May 2017, Pages 379–393.

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]


[edit]
Categories
Parameters
None