Part:BBa_K5036012
Nanog gRNA3 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 .
Dry Lab Characterization
Our dCas-9 system is responsible for YAP-1 expression enhancement. According to our design, after the assembly of dCas-9 domains, the gRNA navigates them to the YAP-1 gene. We have designed 58 different gRNA using CRISPR ON online software tool. Then, we chose the lowest three gRNA off-targeting designs using CRISPR OFF online software tool and tested their stability by RNAfold online software tool. This multi-step approach led us to the best safe gRNA design with minimal off-targeting effect.
gRNA-1 Stability
Mountain plot
(b)Secondary structures
This figure shows that gRNA-1 records Minimal Free Energy (MFE) of -11.10 kcal/mol.
gRNA-2 Stability
Mountain plot
(b)Secondary structures
This figure shows that gRNA-2 records Minimal Free Energy (MFE) of -13.00 kcal/mol .
gRNA-3 Stability
Mountain plot
(b)Secondary structures
This figure shows that gRNA-3 records Minimal Free Energy (MFE) of -13.90 kcal/mol.
Then we have compared between three previous variants to choose the most stable variant
s shown above, gRNA-3 recorded (-13.90 kcal/mol) which is the most stable variant. Among all variants, despite the minimal difference between their MFE, we have chosen the most stable one to reduce the off-targeting effect of our dCas-9 system .
Experimental Characterization
We have done DNA gel electrophoresis to validate the cloning of our gRNA and GFP into dCas9(C)_NLS-Syn-VEGFR-1
This figure illustrates the amplified fragments of our insert gRNA within P5 and GFP within P1 .
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
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