Confirming CRISPR gene editing 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. 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 may cause genomic instability and disrupt the functionality of otherwise normal genes. This can be a major concern when applying CRISPR/Cas9 system to certain types of applications, such as biomedical and clinical.
Next-generation sequencing (NGS), and especially targeted sequencing, has made it possible to provide quality control to CRISPR editing in a cost-effective way. 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.
Paragon Genomics has successfully designed and delivered multiple CRISPR editing assessment panels to researchers at academic institutions and top CRISPR drug discovery companies.
For predicting potential off-target sites, please refer to the following software:
CleanPlex Custom NGS Panels are powered by Paragon Genomics’ CleanPlex Technology – an ultra-high multiplex PCR-based target enrichment technology for next-generation sequencing (NGS). It features a highly advanced proprietary primer design algorithm and an innovative, patented background cleaning chemistry.
Researchers pick targets or genes that they would like to interrogate, and our 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 may help to guide future development of drug targets and design of Treg cell– based immunotherapies.
Prof. Alexander Marson at UCSF
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 or gene editing efficiency issues, this approach results in variable degrees of transformation. A 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.
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