CRISPR QC: CRISPR Genome Editing Technology with NGS
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) is a powerful genome editing technology that is transforming genetics and is associated with the system CRISPR-Cas9. Specifically, genome editing technology makes it possible to mutate, silence, and modify with regions of interest with high levels of precision.
CRISPR coupled with Next-Generation Sequencing (NGS) enables researchers to screen multiple genomic targets at once in a single tube to investigate both on- and off-target edits. As NGS can provide a high level of multiplexing, it can simultaneously analyze multiple target regions or samples, and it has the capability to provide single-base pair resolution, allowing for the detection of specific genetic changes tit the nucleotide level. This makes it a powerful tool for characterizing the genome post-CRISPR.
How To Determine Off-Target Sites
Sometimes CRISPRv can have off-target effects as a result of imperfect binding of Cas9 and the guide RNA to similar genomic sequences that are not the targets intended for editing. To determine off-target sites for CRISPR, there are a variety of methods that can be used, including in silico analysis, genome-wide sequencing, high-throughput assays, amplicon sequencing, and cloning and sanger sequencing.
- In silico analysis uses computation tools based on similarities between the guide RNA and the entire genome. The tools take into account region matches, mismatches, and bulges.
- Genome-wide sequencing analyzes the entire genome of specific regions of interest to identify genetic changes from CRISPR editing.
High-throughput assays such as GUIDE-seq (Genome-wide, Unbiased Identification of DSBs Enabled by sequencing) captures double-stranded breaks induced by Cas9 and the repair outcomes at the cleavage sites.
- Amplicon sequencing identifies off-target effects by targeting specific genomic regions of interest (potential off-target sites computationally determined) and enabling cost-effective deep sequencing.
- Cloning and sanger sequencing involves cloning off-target sites in order to determine if any mutations are present.
Applications of CRISPR Genome Editing Technology
The CRISPR-Cas9 system can be applied to drug discovery and development, animal disease modeling, genetically modified plant technology, biofuel technology, and gene therapy. For example, various research has indicated that CRISPR technology could be applied to specific diseases such as cancers, AIDS, sickle cell, muscular dystrophy, and others.
Confirming CRISPR Knock-Outs with NGS
NGS is a powerful tool for validating CRISPR-Cas9 knock-outs. The targeted genomic region is initially amplified via PCR and then sequenced using NGS. The technology reduces hands-on time for the most cost-effective and sensitive results.
Targeted NGS panels make it possible to confirm not only knock-outs but also knock-ins, edits, and potential off-target effects.
Why Choose CleanPlex NGS Sequencing Panels for Gene Editing Confirmation
Our experts at Paragon Genomics deliver custom assays in a complete kit form for effective confirmation of gene editing, based on the select targets by researchers. This CRISPR off-target analysis will enable researchers to leverage data regarding their off-target effects to refine their CRISPR/Cas9 strategies.
The CleanPlex NGS Sequencing Panels provide a quick turnaround time (less than 4 weeks from design acceptance to shipment) and high performance as a cost-effective sequencing solution.
CRISPR QC: Why Quality Control is Needed in CRISPR Experiments with NGS
Quality control is important for several reasons when it comes to CRISPR experiments, including:
Accuracy of Genetic Changes: Quality control must be established to confidently measure gene editing efficiency and reduce the chance of off-target effects occurring. Otherwise, there is the risk of incorrect results and interpretations.
Experimental Reproducibility: Without the necessary quality controls in place, CRISPR experiments would not be able to be replicated and repeated.
Safety: Quality control is important for maintaining a specific standard of safety for the subjects. This is especially true for CRISPR experiments that could be applied for therapeutic applications.
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