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CRISPR Cas9 System Activity Detection .pdf



Original filename: CRISPR Cas9 System Activity Detection.pdf
Title: CRISPR Cas9 System Activity Detection
Author: http://www.synbio-tech.com/services/crispr-cas9/mammals-knockout/activity-detection/

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CRISPR Cas9 System Activity Detection
In the CRISPR-Cas9 system, different guide RNA (gRNA) can be designed based on
the target gene. The DNA cutting efficiency of Cas9 can vary depending on the
gRNA and target gene. It is therefore important to be able to accurately verify the
activity of the CRISPR-Cas9 system in a given assay, in order to maximize the
cleavage and gene knock-in/knock-out efficiency of CRISPR-Cas9. Synbio
Technologies provides SSA activity assays, in vitro cleavage activity assays, and
endogenous activity assays to detect gRNA activity.

SSA activity assays
Single-strand annealing(SSA)assays can verify whether the target gRNA plasmid
can mediate Cas9 to cut naked DNA and are a common method to assess the
activity of CRISPR-Cas9. The plasmid-based SSA assay contains two inactive
luciferase-encoding fragments, which contain a stop codon and a segment of gRNA
in the middle of the target sequence. If CRISPR-Cas9 can recognize and cleave the
target site, the resultant double-stranded break can be repaired by the SSA
mechanism, rescuing the now-active luciferase gene. Thus the SSA assay can be
used to predict the cleavage activity of CRISPR-Cas9 by fluorescence detection.

Construction of plasmid-based SSA

Contact us
Synbio Technologies
1 Deer Park Drive, Suite L-1
Monmouth Junction
NJ, 08852

Tel:+1 732-230-3003
Fax:+1 609 228 5911
Web:www.synbio-tech.com
E-mail:service@synbio-tech.com

Result of SSA assays
Auxiliary product: construction of plasmid-based SSA activity kit (catalog no.: k0001)

Verification of in vitro cleavage assay
The target DNA sequence is digested with Cas9 in vitro to obtain two DNA
fragments. Agarose gel electrophoresis allows analysis of gRNA-mediated DNA
cleavage by comparing the yield of the smaller, digested fragments to the
remaining undigested target DNA. Verification of in vitro cleavage is another way to
determine the activity and efficiency of CRISPR-Cas9.

Auxiliary product: in vitro gRNA target cleavage activity detection kit (catalog no.: k0002)

Endogenous Activity Assay
Contact us
Synbio Technologies
1 Deer Park Drive, Suite L-1
Monmouth Junction
NJ, 08852

Tel:+1 732-230-3003
Fax:+1 609 228 5911
Web:www.synbio-tech.com
E-mail:service@synbio-tech.com

Surveyor method is called mismatch endonuclease assay, using the endonuclease
T7E1 endonuclease. The suitable primers are first designed on both sides of the
target, after which the DNA containing potentially mismatched mutation sites is
amplified by PCR. T7E1 endonuclease can recognize and cleave mismatched DNA
heteroduplexes. The digested product is finally analyzed by agarose gel
electrophoresis to estimate the ratio of the cleavage and un-cleaved bands,
reflecting the activity of CRISPR-Cas9.

Comparison of different activity assay services:
Assay services

Differentiation

SSA activity assay

Easy to design, simple to use, cost-effective

in vitro

cleavage activity Easy to use, only target design is needed
assay
Cell-free assay system, cost effective

Endogenous activity assay

Fast turnaround time, accelerated validation
process

Service Specifications:
Service Name

Service Specifications


SSA activity
assay




gRNA target primer design and
synthesis
pSSA-luc vector construction
Cell transfection

Timeline Deliverables

Inquiry

Contact us
Synbio Technologies
1 Deer Park Drive, Suite L-1
Monmouth Junction
NJ, 08852

Tel:+1 732-230-3003
Fax:+1 609 228 5911
Web:www.synbio-tech.com
E-mail:service@synbio-tech.com

Assay
Report

in vitro cleavage
activity assay
Endogenous
Activity Assay








In vitro transcription gRNA
CRISPR-Cas9 in
vitro cleavage reaction
Genome extraction
Target primer design
T7E1 enzyme digestion

Inquiry

Assay
Report

Inquiry

Assay
Report

Contact us
Synbio Technologies
1 Deer Park Drive, Suite L-1
Monmouth Junction
NJ, 08852

Tel:+1 732-230-3003
Fax:+1 609 228 5911
Web:www.synbio-tech.com
E-mail:service@synbio-tech.com


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