Determining mechanism of insertational mutagenesis caused by CRISPR/Cas9 genome editing
Lead Research Organisation:
CARDIFF UNIVERSITY
Department Name: School of Medicine
Abstract
Little is currently known about the detailed mechanism of DNA repair initiated by the oligonucleotide-targeted induction of DNA double strand breaks by the CRISPR/Cas9 system. It is currently thought that the doublestrand breaks are repaired by one of the two major double strand break pathways; NHEJ and HR. However, at present, it is not clear what the relative contribution of these two repair pathways in 'on' or 'off'-target CRISPR/Cas9 induced events is. To investigate this we will use novel microarray-based methods developed in Professor Reed's group, for investigating the genomic mechanism of DNA damage induction and its repair in the context of chromatin. This will be combined with the Reed's sophisticated computer algorithms, necessary for the bioinformatics analysis of this data. These technologies have already been developed to investigate the DNA repair mechanisms of genotoxic agents (hence this project will also allow access and validation of these tools for other research applications).
People |
ORCID iD |
| Felix Dobbs (Student) |
Studentship Projects
| Project Reference | Relationship | Related To | Start | End | Student Name |
|---|---|---|---|---|---|
| BB/P504841/1 | 01/10/2016 | 30/03/2021 | |||
| 1803108 | Studentship | BB/P504841/1 | 01/10/2016 | 31/03/2021 | Felix Dobbs |
| Description | We have developed a novel, unbiased, next generation sequencing (NGS) based method to detect DNA double strand breaks (DSBs) throughout the genome called INDUCE-seq. This technique is designed to address the biases and caveats of alternative methods, and measures both the location and frequency of genomic DSBs more accurately than using standard sequencing approaches. Development of this method provides a valuable resource for the measurement of CRSIPR/Cas9 induced on- and off-target breaks in cells, in a manner that is more unbiased and quantitative that any existing techniques. In addition to this, we have established a collaboration with a US-based industrial partner, and benefited from a research project grant to further develop this work. To date, we have demonstrated INDUCE-seq in a range of cell-lines including HEK293, HEK293T, RPE1, U2OS and iPSCs, in addition to in vitro differentiated neural progenitor cells and mature neurones. Furthermore INDUCE-seq can reliably and sensitively detect a range of different DNA damages arising from cell treatment. These include in situ restriction digest by HindIII, live cell treatment using CRSIPR/Cas9, the restriction endonuclease AsiSI, and hydroxyurea (HU). From these initial cell-line experiments, we have demonstrated INDUCE-seq to be reproducible and robust, and have been presented these finding at both National and International conferences. |
| Exploitation Route | The method that we have developed may also be applied to the measure of DSB induced from a variety of sources. These include the measure the activities of alternative genome editing nucleases, use in genotoxicity testing, and the study of genome stability. |
| Sectors | Pharmaceuticals and Medical Biotechnology |
| URL | https://nc3rs.org.uk/induce-seq-novel-tool-next-generation-risk-assessment |
| Description | The development of the novel method INDUCE-seq has led to the formation of a Spin-out company called Broken String Biosciences (Company no. 12420619). In addition to this, our findings have also resulted in a provisional patent application, to facilitate further impact and development of the technology across a broader range of applications. In Feb 2021, Broken String Biosciences was selected to enter the 2nd Global Illumina Accelerator programme, based in Cambridge, UK. Through this opportunity, the company has received pre-seed investment, access to laboratories, and access to Illumina's network of genomics experts and investor community. I am now functioning as CEO of the company. . |
| First Year Of Impact | 2019 |
| Sector | Pharmaceuticals and Medical Biotechnology |
| Impact Types | Economic |
| Title | NGS-based DNA double strand break capture method |
| Description | We have developed a novel method for the detection of DNA double strand breaks in cells or tissue. The method is adapted from and aims to improve on the current state of the art in genome-wide, nucleotide resolution DSB mapping. We have implemented a novel PCR-free enrichment process to improve the sensitivity and availability of the assay, while removing significant PCR amplification biases in the process. |
| Type Of Material | Technology assay or reagent |
| Year Produced | 2018 |
| Provided To Others? | No |
| Impact | This tool may be applied to the study of breaks induced throughout the genome from a variety of sources. It may be used as a tool for the measurement of genotoxicity in human cell samples, which could potentially provide an in vitro substitute for animal models. |
| Company Name | BROKEN STRING BIOSCIENCES LIMITED |
| Description | Broken String Biosciences was formed to take advantage of the novel method INDUCE-seq that was developed during this award. The company aims to provide quantitative and accurate genome-wide DSB measurement to both industry and academic customers for a range of of biomedical applications. |
| Year Established | 2020 |
| Impact | No notable impacts at this time. |