The CRISPR-Quest gene editing test uses XNA technology to screen for the mutants created by CRISPR/CAS 9 gene editing technique. The wildtype gene sequence that covers the expected mutation site is chosen for the custom XNA design. The custom-synthesized XNA then is used in a qPCR reaction to block the wildtype sequence amplification but selectively amplify the DNA containing the mutant sequence. The screening method can be used to screen for mutants from selected pools, or screen/confirm individual clones that contain the desired mutations. The mutant DNA can be further sequenced by Sanger method.
The method is especially effective for high throughput screening for guide RNAs and mutant clones.
Below example shows the XNA design for the WTAP gene. The XNA sequence used for the design, the PAM sequence and cleavage site is shown below.
We would need the information to help you design the primers and XNA for screening the mutants and further sequencing the mutant using Sanger sequencing. Contact us for XNA design used for gene editing mutant screening.
How Does the XNA Screening Work?
In a typical Non-homology End Joining (NHEJ) mutant screening process after CRISPR gene-editing event, One can run an SYBR Green qPCR using a positive control (wild-type DNA plus primers, no XNA), a negative control (wildtype DNA plus primers, with XNA), and the sample DNA (from cell pool or individual clones). The amplification plot for a mutant due to a nucleotide substitution, or an indel, will be located between the plots for positive control and the negative control, as shown be on the right. If there is no mutation present, the sample amplification plot will be similar to the negative control plot.
Based on the Cq value difference (∆Cq) between the Cq for the mutant and the positive control, the percentage of the editing cell proportion can be roughly estimated although it is not accurate.
Editing Events %=2 –∆Cq x100%
For instance, if the Cq for the positive control is 25 cycles and the Cq for the mutant is 27 cycles, the ∆Cq is 2 and the editing events is 25%. However, for mutant screening or gRNA screening, direct comparison of Cq value of each sample with that of the wildtype should give clues which qRNA works better out of the 3 or 4 gRNA used.
We can synthesize the XNA enough for 500 and 1000 reactions for your screening needs. The reactions come with
qPCR mix, one pair of qPCR primers, XNA, positive and negative control. Larger batch of custom synthesis is also available.
CRISPR Makes it Easier for Creating Stable Cell Lines with Particular Gene Mutations
Genome editing by the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system revolutionizes creation of gene mutations. The research community’s rapid acceptance of the CRISPR/Cas9 technology makes gene editing a routine practice for molecular biology labs. Creating gene knockout mutations are unprecedented easy to do for any genes of interest. No more traditional gene knockout and siRNA or shRNA knock-down techniques are necessary.
Challenges for Screening for Desired Mutants from CRISPR Gene Editing
One of the biggest challenges for using the CRISPR system may be the slow turnaround time from introduction of the CRISPR system to identification of the clones with desired mutations due to the low efficiency of current technology by the nature of gene disruption mechanism.
After transfection with the guide RNA (gRNA) and Cas9 system, the cells or cell clones with possible mutations are selected or enriched either with antibiotics selection or with reporter gene expression sorting. The real challenge is to select the low percentage of clones that contain the mutants of interest from most of the clones with wild-type sequences.
PCR followered by restriction enzyme digestions
PCR followed by mismatch assays
High resolution melting analysis
Sanger sequencing combined with PCR
Competition-based PCR method
Utilizing XNA Technology for Mutant Screening
XNA is the Optimal Choice for Screening Mutant clones after CRISPR Gene Editing Compared to other Technologies
Regular PCR methods fail to identify the mutant in
In addition to identify the mutant clones, the technique also enables empirical validation of guide RNA efficiency and measurement of the ratio of HDR: NHEJ at a targeted locus.
Fast to Results
Just set up the qPCR reaction and get results in 3 hours. In contrast, some other methods are tedious and need much more
No Manual Design
We will design the XNA for you if you tell us your wild-type PAM flanking sequence. Order the XNA and qPCR reactions when gRNA is ordered.
Cell lines with low transfection efficiency or poor editing efficiency because of gRNA design often generate low percentage gene editing and needs more clones to screen. XNA technology helps identify low frequent edits in pools where wildtype sequence is dominant. XNA screening also makes high
CRISPR-Quest Gene Editing Test Supporting Data
The rare cell with a single-base change is isolated using XNA technology as a measurement tool, then enriched sequentially until a pure clone resulted.
Calculated Percentage Modification
|CRISPR-Quest BioRad/Taq||CRISPR-Quest NEB||Amplicon Sequencing||T7 Endonucleases|
CRISPR-Quest Gene Editing Test Product Specifications
Product Catalog Number
500-Reaction Pack Size: DC-70-0500R;
1000-Reaction Pack Size: DC-70-1000R
For research use only
500 Reactions and 1000 Reactions
Stable for 12 Months at -25°C to -15°C