Generation and comparison of CRISPR-Cas9 and Cre-mediated genetically engineered mouse models of sarcoma

Abstract

Genetically engineered mouse models that employ site-specific recombinase technology are important tools for cancer research but can be costly and time-consuming. The CRISPR-Cas9 system has been adapted to generate autochthonous tumours in mice, but how these tumours compare to tumours generated by conventional recombinase technology remains to be fully explored. Here we use CRISPR-Cas9 to generate multiple subtypes of primary sarcomas efficiently in wild type and genetically engineered mice. These data demonstrate that CRISPR-Cas9 can be used to generate multiple subtypes of soft tissue sarcomas in mice. Primary sarcomas generated with CRISPR-Cas9 and Cre recombinase technology had similar histology, growth kinetics, copy number variation and mutational load as assessed by whole exome sequencing. These results show that sarcomas generated with CRISPR-Cas9 technology are similar to sarcomas generated with conventional modelling techniques and suggest that CRISPR-Cas9 can be used to more rapidly generate genotypically and phenotypically similar cancers.

Injury promotes sarcoma development in a genetically and temporally restricted manner

“…10). The mutational frequency in the Kras-induced sarcomas was very similar to sarcomas that arise in Trp53 fl/fl ; Kras LSL-G12D mice treated with IM adeno-Cre (11).…”

“…Assessment of Kras recombination. Kras recombination in sarcoma cell lines was assessed using a PCRbased assay as previously described (11).…”

“…Exome sequencing. Preparation and analysis of DNA was performed in an identical fashion as we have recently described (11). In brief, sequencing utilized the Agilent Mouse All Exon kit, followed by high-throughput sequencing on the Illumina HiSeq 2500 platform.…”

CRISPR-mediated modeling and functional validation of candidate tumor suppressor genes in small cell lung cancer

“…However, we also observed several differences in tumor phenotype between p107-mutated and p130mutated tumors, indicating potential biological differences between SCLC tumors with these genotypes. CRISPR-mediated knockout of genes has been particularly useful for modeling loss-of-function mutations in tumor suppressor genes, such as those as demonstrated here, as well as in other models (25,27,30,32). Other studies have demonstrated the ability to generate gain-of-function alterations, such as chromosomal rearrangements (26), as well as homologous recombinationmediated activation of oncogenes (25), albeit at a very low efficiency in the latter case.…”

“…The development of the CRISPR-Cas9 system for genome editing in mammalian cells (22)(23)(24) has revolutionized the field of cancer research, enabling rapid validation of candidate oncogenes and tumor suppressor genes both in vitro as well as in vivo. This has been especially useful when combined with GEMMs of various cancers (25)(26)(27)(28)(29)(30)(31)(32). By bypassing the need to generate new germline or conditional alleles for each gene of interest, the CRISPR-Cas9 Significance SCLC is a deadly disease for which treatment outcomes have not improved significantly for over 30 y due to the lack of effective new therapies.…”