Part:BBa_K5036040

CRISPR multiplexing system (Nanog gRNA-HHR-gRNA operator)

Part Description

CRISPR multiplexing system is a versatile genetic engineering tool, that empowers scientists to simultaneously target and modify multiple genes within an organism. By designing multiple guide RNAs that precisely bind to specific DNA sequences of interest, researchers can efficiently and accurately introduce desired genetic changes. This capability enhances efficiency and precision, making CRISPR a valuable asset for a wide range of biological applications.

Usage

Our CRISPR multiplexing system consists of three components: a guide RNA targeting the Nanog gene to activate endogenous YAP-1 production by (our dCAS9 transcription activator) complex, a guide RNA targeting a sequence to activate the production of mRNA containing the TID switch by (our dCAS9 transcription activator) complex, and the HHR enzyme which mediates self-cleavage to separate these guide RNAs into individual functional units.

this figure illustrates the process our CRISPER multiplexing system which Allow us to fully utilize the capabilities of our dCas9(N/C)-TF-SynVEGFR1/2 system .

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]