CRISPR-Barcoding for Intratumor Genetic Heterogeneity Modeling and Functional Analysis of Oncogenic Driver Mutations

Abstract

SUMMARY Intratumor genetic heterogeneity underlies the ability of tumors to evolve and adapt to different environmental conditions. Using CRISPR/Cas9 technology and specific DNA barcodes, we devised a strategy to recapitulate and trace the emergence of subpopulations of cancer cells containing a mutation of interest. We used this approach to model different mechanisms of lung cancer cell resistance to EGFR inhibitors and to assess effects of combined drug therapies. By overcoming intrinsic limitations of current approaches, CRISPR-barcoding also enables investigation of most types of genetic modifications, including repair of oncogenic driver mutations. Finally, we used highly complex barcodes inserted at a specific genome location as a means of simultaneously tracing the fates of many thousands of genetically labeled cancer cells. CRISPR-barcoding is a straightforward and highly flexible method that should greatly facilitate the functional investigation of specific mutations, in a context that closely mimics the complexity of cancer.

Progress and Application of CRISPR/Cas Technology in Biological and Biomedical Investigation

“…Now combined with the gene editing functionality of CRISPR/Cas9, it has been adopted to trace lineage differentiation of interested cell line in vitro or in vivo. Guernet et al [,] developed a CRISPR‐barcoding strategy in studying of intratumor heterogeneity. They induced qPCR‐readable DNA barcodes in lung cancer cell line using CRISPR, and traced the emergence and fate of subpopulations resistant to EGFR inhibitor and investigated the intrinsic cells interactions and mutated genes.…”

CRISPR-barcoding in non small cell lung cancer: from intratumor genetic heterogeneity modeling to cancer therapy application

“…They generated models of NSCLC resistance to EGFR inhibitors based on a specific sgRNA and a donor singlestranded DNA oligonucleotide (ssODN) containing as barcodes different genetic aberrations, including the EGFR T790M mutation, a secondary mutation in the catalytic domain associated to acquired resistance and KRAS G12D mutation, a well-known negative predictor for primary responsiveness to EGFR inhibitors. Studies in immunocompromised mice injected with KRAS-G12D and EGFR-T790M CRISPR-barcoded cells showed an increase of both mutations in the tumors from gefitinib treated mice, demonstrating that resistant subclones were selected (6).…”

“…NHEJ frequently results in the insertion or deletion of a few nucleotides and it can be used to knockout the gene of interest thought frameshift mutations. Repair by HDR include a donor DNA template displaying sequence homology to the targeted locus and it can be exploited to modify a gene by introducing point mutations or to insert more extensive modifications achieve of the genome (6).…”

“…This new technology enables a highresolution tracking of single specific cancer cells allowing to identify even rare pre-existing resistant subclones potentially involved in mechanisms of acquired resistance to therapy (6 Guernet et al highlighted particularly the use of CRISPR-barcoding to develop models of drug resistance in non-small cell lung cancer (NSCLC) since this system closely mimics the clonal dynamics of cancer. They generated models of NSCLC resistance to EGFR inhibitors based on a specific sgRNA and a donor singlestranded DNA oligonucleotide (ssODN) containing as barcodes different genetic aberrations, including the EGFR T790M mutation, a secondary mutation in the catalytic domain associated to acquired resistance and KRAS G12D mutation, a well-known negative predictor for primary responsiveness to EGFR inhibitors.…”

Modeling intratumor heterogeneity through CRISPR-barcodes

“…We recently devised a novel approach based on new technologies for DNA editing to recapitulate and trace the emergence of a new mutation in a subset of cancer cells, thus enabling functional studies on a gene of interest in a context that mimics intratumor heterogeneity. 3 Originally an adaptive immune system in prokaryotes, CRISPR (clustered regularly interspaced short palindromic repeats) has been engineered into a new powerful tool for genome editing. 4,5 This system is composed of the Cas9 nuclease from S. Pyogenes and a short RNA sequence, the singleguide RNA (sgRNA).…”