CRISPR Editing QC

Confirming CRISPR gene editing knockouts / knock-ins and evaluating off-target effects

CRISPR Editing QC

Concerns when applying CRISPR/Cas9 system

  • CRISPR/Cas 9 system is an amazing tool that can be used for an applications of gene editing such as for genetically modified plants, animal disease modelling, gene therapy etc.

  • Using CRISPR/Cas 9 tool, we can either carry out gene silencing or gene editing (addition of new gene) .

  • QC of CRISPR is necessary for confirmation of knockout / knock-in and off-target effect evaluation.

  • 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 can cause genetic instability and disrupt the functionality of normal genes

Confirming CRISPR Gene Editing

  • Next generation sequencing is simple and cost effective method for CRISPR QC.

 

  • 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. 

  • Researchers pick targets or genes that they would like to interrogate, and our experts design and deliver the custom assays in complete kit format

  • 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.

Our Solution for CRISPR QC

Case Study

One of the successful CRISPR panel examples is Professor Marson’s lab at UCSF using a CleanPlex custom panel for CRISPR / gene editing assessment. Prof. 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 CleanPlex custom panels of Paragon Genomics, 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.

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