Somatic genome editing with the RCAS-TVA-CRISPR-Cas9 system for precision tumor modeling

Abstract

To accurately recapitulate the heterogeneity of human diseases, animal models require to recreate multiple complex genetic alterations. Here, we combine the RCAS-TVA system with the CRISPR-Cas9 genome editing tools for precise modeling of human tumors. We show that somatic deletion in neural stem cells of a variety of known tumor suppressor genes (Trp53, Cdkn2a, and Pten) leads to high-grade glioma formation. Moreover, by simultaneous delivery of pairs of guide RNAs we generate different gene fusions with oncogenic potential, either by chromosomal deletion (Bcan-Ntrk1) or by chromosomal translocation (Myb-Qk). Lastly, using homology-directed-repair, we also produce tumors carrying the homologous mutation to human BRAF V600E, frequently identified in a variety of tumors, including different types of gliomas. In summary, we have developed an extremely versatile mouse model for in vivo somatic genome editing, that will elicit the generation of more accurate cancer models particularly appropriate for pre-clinical testing.

MGMT genomic rearrangements contribute to chemotherapy resistance in gliomas

“…To characterize the MGMT fusions, we sought to generate some of the identified rearrangements using the CRISPR/Cas9-mediated genome editing. Co-expression of Cas9 with pairs of single-guide RNAs (sgRNAs) has been used to model a variety of chromosomal rearrangements (such as translocations, inversions, and deletions) by creating DNA double-strand breaks at the breakpoints of chromosome rearrangements, which are then joined by non-homologous end joining 10 , 11 . To generate cell lines carrying the MGMT fusions, we first transduced U251 and U87 cells, two MGMT-methylated GBM cell lines, with lentiviral vectors expressing different combinations of gRNA pairs directed to four different MGMT rearrangements: BTRC-MGMT , NFYC-MGMT , SAR1A-MGMT , and CTBP2-MGMT (Supplementary Fig.…”

“…The gRNA sequences targeting MGMT, BTRC, NFYC, SAR1A, and CTBP2 were designed using the Genetic Perturbation Platform web portal ( http://portals.broadinstitute.org/gpp/public/analysis-tools/gRNA-design ) (Supplementary Table 3 ). The paired sgRNAs were sub-cloned into the pKLV-U6gRNA-PGKpuro2ABFP, as previously described 11 . Briefly, the oligonucleotides containing the different gRNA pairs (Supplementary Table 4 ) were amplified with Phusion High-Fidelity polymerase (New England Biolabs, M0530S) using primer F5 and R1 (Supplementary Table 2 ).…”

“…Genomic DNA was isolated as previously described 11 . Briefly, cell pellets were incubated in lysis buffer (10 mM Tris-HCl ph8, 100 mM NaCl, 0.5 mM EDTA, 10% SDS, and proteinase K) for 4 h at 55 °C, and genomic DNA was extracted using phenol:chloroform (1:1) and Phase Lock heavy 2-ml tubes (5PRIME, Cat.…”

Drug Inducible CRISPR/Cas Systems

“…In addition, Cre activity can be regulated by fusing with the ligand binding domain (LBD) of the ER or its mutant, rendering a tamoxifen-dependent Cre action [27–29], or by fusing with the LBD of a mutated progesterone receptor which responds to the synthetic steroid RU486 but not endogenous progesterone [[30], [31]]. These drug inducible Cre systems have been used to control Cas9 activity through recombination of the LSL cassette [[32], [33]].…”

Next-Generation in vivo Modeling of Human Cancers

“…This report by the Ventura lab builds on their own pioneering work in generating an EML4-ALK fusion model for NSCLC ( 60 ), using CRISPR/Cas9 to systematically screen for fusions as oncogenic drivers in solid tumors. Using this approach, in addition to the chromosomal deletion required for the Bcan - Ntrk1 model, it has been possible to model the Myb - Qk chromosomal translocation as well as induce an equivalent of the human BRAF V600E point mutation by homology-directed-repair ( 52 ). The latter improvements in genetic engineering now make easier to generate more complex genotypes in autochthonous models.…”