Part:BBa_K5036035

U6 promoter- Nanog gRNA1 (scaffold- spacer RNA )

Part Description

This part consists of the U6 promoter, which constantly activates the creation of our Nanog guide RNA, and The Nanog guide RNA comprises a spacer sequence that matches the Nanog DNA sequence, enabling gene editing. Additionally, the guide RNA includes a scaffold sequence that binds to the dCas9 protein, allowing it to interact with the target DNA

Usage

Our guide RNA is used with NLS to direct dCas9 linked to transcription activators VP64,UAS TransCMV enhancer and GAL4 into the nucleus to target Nanog gene which is located upstream to YAP-1 coding region which induce transcription and increase production of endogenous YAP-1.

This figure illustrates the mechanism of action of Nanog gRNA in our model.

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 .

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
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 316
  
• 1000
  COMPATIBLE WITH RFC[1000]