A systematic genome-wide mapping of oncogenic mutation selection during CRISPR-Cas9 genome editing (original) (raw)
Related papers
Unsupervised correction of gene-independent cell responses to CRISPR-Cas9 targeting
BMC Genomics, 2018
Background: Genome editing by CRISPR-Cas9 technology allows large-scale screening of gene essentiality in cancer. A confounding factor when interpreting CRISPR-Cas9 screens is the high false-positive rate in detecting essential genes within copy number amplified regions of the genome. We have developed the computational tool CRISPRcleanR which is capable of identifying and correcting gene-independent responses to CRISPR-Cas9 targeting. CRISPRcleanR uses an unsupervised approach based on the segmentation of single-guide RNA fold change values across the genome, without making any assumption about the copy number status of the targeted genes. Results: Applying our method to existing and newly generated genome-wide essentiality profiles from 15 cancer cell lines, we demonstrate that CRISPRcleanR reduces false positives when calling essential genes, correcting biases within and outside of amplified regions, while maintaining true positive rates. Established cancer dependencies and essentiality signals of amplified cancer driver genes are detectable post-correction. CRISPRcleanR reports sgRNA fold changes and normalised read counts, is therefore compatible with downstream analysis tools, and works with multiple sgRNA libraries. Conclusions: CRISPRcleanR is a versatile open-source tool for the analysis of CRISPR-Cas9 knockout screens to identify essential genes.
Identification of oncogenic driver mutations by genome-wide CRISPR-Cas9 dropout screening
BMC genomics, 2016
Genome-wide CRISPR-Cas9 dropout screens can identify genes whose knockout affects cell viability. Recent CRISPR screens detected thousands of essential genes required for cellular survival and key cellular processes; however discovering novel lineage-specific genetic dependencies from the many hits still remains a challenge. To assess whether CRISPR-Cas9 dropout screens can help identify cancer dependencies, we screened two human cancer cell lines carrying known and distinct oncogenic mutations using a genome-wide sgRNA library. We found that the gRNA targeting the driver mutation EGFR was one of the highest-ranking candidates in the EGFR-mutant HCC-827 lung adenocarcinoma cell line. Likewise, sgRNAs for NRAS and MAP2K1 (MEK1), a downstream kinase of mutant NRAS, were identified among the top hits in the NRAS-mutant neuroblastoma cell line CHP-212. Depletion of these genes targeted by the sgRNAs strongly correlated with the sensitivity to specific kinase inhibitors of the EGFR or RA...
Science Advances
Most genome editing analyses to date are based on quantifying small insertions and deletions. Here, we show that CRISPR-Cas9 genome editing can induce large gene modifications, such as deletions, insertions, and complex local rearrangements in different primary cells and cell lines. We analyzed large deletion events in hematopoietic stem and progenitor cells (HSPCs) using different methods, including clonal genotyping, droplet digital polymerase chain reaction, single-molecule real-time sequencing with unique molecular identifier, and long-amplicon sequencing assay. Our results show that large deletions of up to several thousand bases occur with high frequencies at the Cas9 on-target cut sites on the HBB (11.7 to 35.4%), HBG (14.3%), and BCL11A (13.2%) genes in HSPCs and the PD-1 (15.2%) gene in T cells. Our findings have important implications to advancing genome editing technologies for treating human diseases, because unintended large gene modifications may persist, thus altering...
Genomic copy number dictates a gene-independent cell response to CRISPR-Cas9 targeting
Cancer discovery, 2016
The CRISPR-Cas9 system enables genome editing and somatic cell genetic screens in mammalian cells. We performed genome scale loss-of-function screens in 33 cancer cell lines to identify genes essential for proliferation/survival and found a strong correlation between increased gene copy number and decreased cell viability after genome editing. Within regions of copy number gain, CRISPR-Cas9 targeting of both expressed and unexpressed genes, as well as intergenic loci, led to significantly decreased cell proliferation through induction of a G2 cell cycle arrest. By examining single guide RNAs that map to multiple genomic sites, we found that this cell response to CRISPR-Cas9 editing correlated strongly with the number of target loci. These observations indicate that genome targeting by CRISPR-Cas9 elicits a gene-independent anti-proliferative cell response. This effect has important practical implications for interpretation of CRISPR-Cas9 screening data and confounds the use of this ...
CRISPR-Cas9: a promising genetic engineering approach in cancer research
Therapeutic advances in medical oncology, 2018
Bacteria and archaea possess adaptive immunity against foreign genetic materials through clustered regularly interspaced short palindromic repeat (CRISPR) systems. The discovery of this intriguing bacterial system heralded a revolutionary change in the field of medical science. The CRISPR and CRISPR-associated protein 9 (Cas9) based molecular mechanism has been applied to genome editing. This CRISPR-Cas9 technique is now able to mediate precise genetic corrections or disruptions inandenvironments. The accuracy and versatility of CRISPR-Cas have been capitalized upon in biological and medical research and bring new hope to cancer research. Cancer involves complex alterations and multiple mutations, translocations and chromosomal losses and gains. The ability to identify and correct such mutations is an important goal in cancer treatment. In the context of this complex cancer genomic landscape, there is a need for a simple and flexible genetic tool that can easily identify functional ...
Cancer discovery, 2016
CRISPR/Cas9 has emerged as a powerful new tool to systematically probe gene function. In this study, we compare the performance of CRISPR to RNAi-based loss-of-function screens for the identification of cancer dependencies by performing parallel deep-coverage shRNA and CRISPR screens targeting 2722 genes across several cancer cell lines. CRISPR-based dropout screens identified more lethal genes compared to RNAi in all five cancer models, indicating that the identification of many cellular dependencies may require full gene inactivation, as induced by CRISPR but not RNAi. However, in two aneuploid cancer models we found that all genes within highly amplified regions, including non-expressed genes, scored as lethal by CRISPR, revealing an unanticipated class of false-positive hits in CRISPR-based screens. Using a CRISPR tiling array that encompassed all possible sgRNAs against the coding regions of 139 genes, we found that sgRNAs targeting essential domains provide the most robust dro...
2017
The CRISPR-Cas9 system has revolutionized gene editing both on single genes and in multiplexed loss-of-function screens, enabling precise genome-scale identification of genes essential to proliferation and survival of cancer cells. However, previous studies reported that an anti-proliferative effect of Cas9-mediated DNA cleavage confounds such measurement of genetic dependency, particularly in the setting of copy number gain1-4. We performed genome-scale CRISPR-Cas9 essentiality screens on 342 cancer cell lines and found that this effect is common to all lines, leading to false positive results when targeting genes in copy number amplified regions. We developed CERES, a computational method to estimate gene dependency levels from CRISPR-Cas9 essentiality screens while accounting for the copy-number-specific effect, as well as variable sgRNA activity. We applied CERES to sets of screens performed with different sgRNA libraries and found that it reduces false positive results and prov...
Frontiers of CRISPR-Cas9 for Cancer Research and Therapy
Journal of Exploratory Research in Pharmacology, 2021
In recent years, gene editing technologies have made significant progress in understanding gene function and regulation. The Clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9) system has emerged as a versatile tool for gene editing and genome engineering. In the last few years, CRISPR-Cas9 technology has been widely applied to cancer research, mainly to understand the mechanisms of oncogenesis, drug-target identification, and the development of various cell-based therapies. When combined with genome sequence information, this technology has also shown promise to cure heritable genetic disorders. This review summarizes some of the recent developments and preclinical applications of CRISPR-Cas9 technology in cancer research and therapy. We will discuss how CRISPR based approaches have been used as a tool to identify cancer-specific vulnerabilities and potential applications in cancer therapy.