Confirming CRISPR Gene Editing Confirmation Knockouts / Knock-Ins and Evaluating Off-Target Effects

The CRISPR-Cas9 system holds great promise in diverse fields such as drug discovery and development, animal disease modeling, genetically modified plant technology, biofuel technology, and gene therapy. As gene editing via CRISPR becomes a popular tool, the need for CRISPR quality control, knockout / knock-in confirmation and off-target effect evaluation is also increasing.

Generally speaking, after Cas9 has created, a double-stranded DNA break at the target region, DNA repair starts via the non-homologous end joining pathways (NHEJ) or homology-directed repair (HDR) that result in creation of short insertions and deletions (INDELS). The indels can result in a frameshift mutation for all copies of the targeted gene, which leads to a gene knockout. Due to editing efficiency issues, CRISPR editing often results in a mixed cell population with only a small percentage of the total cells edited successfully. Off-target mutations observed at frequencies similar to or greater than the intended mutations, which might cause genomic instability and disrupt the functionality of otherwise normal genes, are still one major concern when applying CRISPR/Cas9 system to biomedical and clinical applications. For example, when targeting one gene of a diploid cell, there are four possible outcomes, 1. no gene is edited, 2. heterozygous mutation (only 1 allele is edited), 3. biallelic mutation (both allele are edited but they carris 2 different mutations), 4. homozygous mutation. In most cases, both biallelic and homozygous mutations are desired to be sure that the gene of interest is knocked out.  Gene knockout in multiple locations complicats the validation of gene editing, therefore, choosing the right tool to confirm CRISPR gene editing is essential.

Polyclonal screening can be done by amplicon based PCR following by Sanger sequencing, however, polyclonal screening are expensive and complicated to analyze even though it provides general indel sequence information.  In Paragon Genomics, we do large scale of monoclonal screening using next generation sequencing (NGS) by designing a targeted NGS panel covering the intended mutation sites and possible off-target sites, sequencing results can confirm knockouts, knock-ins, edits, and potential off-target effects. Library preparation for NGS typically relies on enzymatic reactions, such as fragmentation with restriction enzymes or sonication, followed by the addition of adapters and PCR amplification steps. These steps are aimed at generating a library of DNA fragments that can be sequenced on platforms like Illumina or Oxford Nanopore Technology. Paragon Genomics has successfully designed and delivered multiple CRISPR editing assessment panels to researchers at academic institutions and top CRISPR drug discovery companies.

Product Highlight

CleanPlex Custom NGS Panels are powered by Paragon Genomics’ CleanPlex Technology, an ultra-high PCR amplicon-based targeted sequencing technology for NGS. As mentioned, after researchers select their targets, our experts deliver the custom assay in complete kit format for effective confirmation of gene editing. This CRISPR off-target analysis will enable researchers to leverage data regarding their off-target effects to refine their CRISPR/Cas9 strategies.

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CRISPR Gene Editing with CleanPlex Custom NGS Panels

Researchers pick targets or genes that they would like to interrogate, and the Paragon Genomics’ experts design and deliver the custom assays in complete kit format (input to sequencing-ready NGS libraries) in just 4 to 6 weeks. One of the successful CRISPR panel examples is Professor Marson’s lab at UCSF using a CleanPlex custom panel for CRISPR / gene editing assessment. Professor Alexander Marson in collaboration with researchers from University of California, Berkeley, CA, USA, Technische Universität München (TUM), Munich, Germany, and multiple renowned Cancer Immunotherapy institutes in San Francisco published findings in Nature Immunology showing CRISPR–Cas9 ribonucleoprotein (RNP) technology allows dissection of complex gene modules in primary human Treg cells through targeted gene perturbation studies.

Using our CleanPlex custom panels, researchers were able to perform CRISPR editing efficiency check and show pooled Cas9 RNP screens link indels with phenotypic changes in human Treg cells. The overall study resulted in functional network maps which might help to guide future development of drug targets and the design of Treg cell-based immunotherapies.

Site-Directed Mutagenesis for Mouse Models: Gene Editing QC Panel

Site-directed mutagenesis has emerged as a valuable tool to evaluate how specific alternations in genes affect protein activity. With the rapid advancement in this field, researchers can address both the role of a specific sequence at both a cellular level in vitro and also at a macro level, evaluating the entirety of the organism in vivo. Several PCR-based methods exist to introduce nucleotide changes but often, due to specificity of gene editing efficiency issues, this approach results in variable degrees of transformation. Fast and reliable confirmation of mutagenesis has been a significant challenge in this field.

Paragon Genomics has leveraged its CleanPlex Sequencing Technology to design and deliver an easy targeted editing assessment panel to confirm successful mutagenesis. Each panel is custom-designed for the researcher’s gene of interest with a rapid turn-around-time for design.

Why Choose CleanPlex NGS Sequencing Panels for Gene Editing Confirmation

As mentioned, after researchers select their targets, our experts deliver the custom assay in complete kit format for effective confirmation of gene editing. This CRISPR off-target analysis will enable researchers to leverage data regarding their off-target effects to refine their CRISPR/Cas9 strategies.

With Paragon Genomics CleanPlex NGS Sequencing Panels, you get:

Quick Turnaround Time (TAT):

Accelerate your CRISPR gene editing assay development project and get your personalized NGS panels in less than 4 weeks (from design acceptance to shipment). Easily build a CleanPlex Custom NGS or Sequencing Panel to accelerate your research and assay development.

Use ParagonDesigner™, our free online multiplex PCR primer design tool, to submit your target regions of interest and instantly receive a design coverage report to review. Our custom amplicon panel design experts will be available through the process to provide you a quote, help you with any questions, and make further optimization to meet your needs.

High Performance:

Jumpstart your CRISPR gene editing confirmation work with NGS panels tailored to your needs. Custom panels are expertly designed with high first-pass success and a high target design rate due to Paragon’s highly advanced proprietary primer design algorithm and an innovative, patented background cleaning chemistry. Together, they allow CleanPlex Custom NGS Amplicon Panels to break the limits of existing target enrichment technologies.

Cost-Effective Sequencing for Gene Editing:

Generate CRISPR data with high on-target performance and coverage uniformity to minimize the cost of sequencing.

Ultra-High Multiplexing:

Multiplex up to 20,000+ amplicons per pool for large NGS panels to help you unlock new applications using a faster and simpler workflow for your gene editing confirmation.

Flexible and Scalable Content:

Update your custom NGS Panels as you gather new genome editing confirmation insights.

High Level of Support for Your Gene Editing Confirmation:

Receive speedy and comprehensive support throughout the design and ordering process. Our PhD-level expert scientists are here to help create a custom assay for your genome editing confirmation.

What’s Included in My CRISPR Gene Editing Confirmation CleanPlex Custom NGS Panel?

Each CleanPlex Custom NGS Panel contains all reagents necessary to construct target-enriched, sequencing-ready NGS amplicon libraries from input DNA. Your CleanPlex Custom NGS Panel will include a CleanPlex Targeted Library Kit, your custom multiplex PCR primer mix, CleanMag® Magnetic Beads for bead purification, and Illumina® or Ion Torrent™ index primers for multiplexing and sequencing on the platform of your choice.

For predicting potential off-target sites, please refer to the following software:

CCTOP: Off-target prediction software

Deep neural networks for off-target prediction

COSMID: A Web-based Tool for Identifying and Validating CRISPR/Cas Off-target Sites

Custom NGS Panels for Gene Editing Confirmation: FAQs

Researchers currently use a spectrum of methods to assess on-target efficiency including PCR, Sanger sequencing, cleavage assays, and NGS. However, NGS is the only method that enables multiplexed high characterization and single base pair resolution of the genome after a CRISPR editing experiment. 

In general, an amplicon-sequencing based approach is ideal as it can screen multiple genomic targets at once in a single reaction tube to investigate both on- and off-target edits.

It can evaluate and quantify the frequency of indels and base changes created from CRISPR-generated double-strand breaks and gives a high-resolution picture of editing events at the target site. In terms of process, compared to hybridization capture or another NGS method, it is the least laborious in hands-on-time and effort and the most cost-efficient.

Please submit your design request to the ParagonDeisgner Portal with your targets of interest: regions where you expect intended edits and potential off-target sites and our design team will respond shortly with a panel design to maximize target uniformity and specificity and mitigate any background product that might be generated.

For standard CleanPlex technology, we have designed and delivered panels of up to 20,000 amplicons (approx. 400-genes) with great performance. Panels with higher amplicon targets may require more primer pools, which in turn require more DNA. If the total sample quantity is limited, there could be a cap on how many targets can be assayed.
For CleanPlex UMI technology, sequencing depth is the limiting factor for how many amplicon targets to include in your custom panel. UMI panels require deeper sequencing to fully utilize the resolving power of the technology. The sequencing reads required per sample is calculated based on how low the allele frequencies percentage you wish to detect, how much DNA input you need, and how many amplicons per panel.

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