Gene Knockout Validation

CRISPR/Cas9 is the third generation of gene editing technology following Zinc Finger Nuclease (ZFN) and Transcription-Activator Like Effector Nucleases (TALEN). This technology has been developing rapidly since 2013. In the CRISPR Cas9 system, a sgRNA directs the Cas9 endonuclease to the target loci and then cleave the target gene. Since the gene editating efficiency of the gRNA is unpredictable, therefore, the choice of gRNA with high efficiency and specificity is critical to successful gene editating. For the efficient gRNA screening, researchers typically test multiple gRNAs(3 to 5) per target gene.

WZ Biosciences can provide personalized Gene Kockout Validation Services for you according to your research needs, including Custom gRNA Validation and efficient gRNA Validation.

*Interested in above Kockout Validation Services? Please feel free to contact us to“Request a quote”.

Related services: CRISPR/Cas9 Cloning, Adenovirus Packaging, Lentivirus Packaging, AAV Packaging, Stable Cell Line Generation

In CRISPR/Cas9 experiments, researchers often use sequencing methods to confirm gene edits, including sanger sequencing of PCR products(qualitative) and sanger sequencing of cloned inserts(quantitative). Even though the results of these two methods are reliable, they have several limits, such as complex operation, long-term experiment, cell types limitation, and so on. To address these problems, WZ Biosciences have developed a simple validation system-Fluorescence Method.

Advantages:

Multiple gRNAs targeting some gene are designed and construted. Then we co-transfect the pKO-Reporter plasmids and gRNA & Cas9 expression plasmids into HEK293 cells. Validation of shRNA knockdown efficiency was assessed by the GFP reporter gene. Images below were taken 48h post-transfection.

The chart below shows the comparsion between sanger sequencing of PCR products, sanger sequencing of cloned inserts and Fluorescence Method:

Service Process

Related Resorces

	《Transfection Reagent Instructions》

TALEN Transcription Activator-like Effector Nucleases (TALENs) are artificial restriction enzyme that can cut specific sequences of DNA. TALENs are designed by fusing a TAL effector DNA-binding domain to a DNA cleavage domain (a nuclease), which cuts DNA strands. TALENs introduce a site-specific double-strand break (DSB), which is subsequently repaired by the endogenous DNA repair machinery, such as non-homologous end-joining (NHEJ) and homology-directed repair (HDR), which often creates short DNA insertions or deletions (indels) . The principle is shown in the figure below: TALEs (TAL effectors，Transcription activator-like effectors) The TAL effector (TALE) is natural protein secreted by plant pathogenic bacteria Xanthomonas sp., which can recognize specific DNA base pairs. Design Strategy Advantages: 1.A complete collection of repeat tetramers for assembling 16-20 repeat units directly； 2.Various virus vectors available； 3. Both clones are Fully sequenced； 4. Functional validation available。 Application example---- Integration site AAVS1 gene editing in human genome by AAV transfection Two(Left & Right) AAVS1-TALEN were created using WZ Biosciences’ Custom TALEN Design(Fig.A). AAVS1-TALEN was then sequenced to confirm for TALEN cut outcom(Fig.B & C). Results: 15 clones were sequenced, 9 contains deletions with 8 being different deletions, results in 8 mutations created by the TALEN design.

Comparison of TANLEN & CRISPR/Cas9

Property	TALEN	CRISPR-CAS9
Type of recognition	Protein-DNA	RNA-DNA
Modularity	One RVD recognizes one nucletide	one nucletide to one nucletide
Target sequences	2×16 nucletide and up	~20 nucletide
Off-target effect	Less observed off-target effects	More potential off-target effects
Multiplexing	Rarely used	Capable
Methylation sensitive	Sensitive	Not sensitive