CRISPR/Cas9 somatic multiplex-mutagenesis for high-throughput functional cancer genomics in mice

Abstract

Here, we show CRISPR/Cas9-based targeted somatic multiplexmutagenesis and its application for high-throughput analysis of gene function in mice. Using hepatic single guide RNA (sgRNA) delivery, we targeted large gene sets to induce hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). We observed Darwinian selection of target genes, which suppress tumorigenesis in the respective cellular/tissue context, such as Pten or Cdkn2a, and conversely found low frequency of Brca1/2 alterations, explaining mutational spectra in human ICC/HCC. Our studies show that multiplexed CRISPR/Cas9 can be used for recessive genetic screening or high-throughput cancer gene validation in mice. The analysis of CRISPR/Cas9-induced tumors provided support for a major role of chromatin modifiers in hepatobiliary tumorigenesis, including that of ARID family proteins, which have recently been reported to be mutated in ICC/HCC. We have also comprehensively characterized the frequency and size of chromosomal alterations induced by combinatorial sgRNA delivery and describe related limitations of CRISPR/Cas9 multiplexing, as well as opportunities for chromosome engineering in the context of hepatobiliary tumorigenesis. Our study describes novel approaches to model and study cancer in a high-throughput multiplexed format that will facilitate the functional annotation of cancer genomes.in vivo CRISPR/Cas9 | somatic multiplex-mutagenesis | hepatocellular carcinoma | intrahepatic cholangiocarcinoma | chromosome engineering F or decades, a major bottleneck in cancer research has been our limited ability to identify genetic alterations in cancer. The revolution in array-based and sequencing technologies and the recent development of insertional mutagenesis tools in animal models enable the discovery of cancer-associated genetic alterations on a genome-wide scale in a high-throughput manner. Nextgeneration sequencing (NGS) of cancer genomes and transposonbased genetic screening in mice, for example, are currently creating large catalogs of putative cancer genes for principally all cancer types (1-3). A challenge for the next decades will be to validate the causative cancer relevance of these large gene sets (to distinguish drivers from passengers) and to understand their biological function. Moreover, pinpointing downstream targets of mutated cancer genes or drivers among the thousands of transcriptionally or epigenetically dysregulated genes within individual cancers is complex and limited by the lack of tools for high-throughput functional cancer genomic analyses.The development of technologies for targeted manipulation of the mouse germ line has opened tremendous opportunities to study gene function (4, 5). Mouse models recapitulate the extensive biological complexity of human cancer and have given insights into many fundamental aspects of the disease that can be studied only at an organismal level (6). However, the speed and efficiency of such studies is limited by the long time frames needed to genetically engineer, intercross,...

piggyBac mediates efficient in vivo CRISPR library screening for tumorigenesis in mice

“…A recent CRISPR validation study showed that gene Cdkn2a sgRNA and Ras oncogene overexpression, with sgRNAs targeting nine other tumor suppressor genes (TSGs) delivered by SB transposon, generated tumors, but only at 20-30 wk after injection (15). We performed tail vein injections to test whether Cdkn2a-sgRNA combined with oncogene NRAS G12V overexpression delivered by the PB vectors, pCRISPR-W9-Cdkn2a-sgRNA and pPB-hNRAS G12V (Fig.…”

“…However, because of their low efficiency, these two methods have not been widely used. Recently, CRISPR/Cas9 has been developed as an efficient mutagenesis tool (7)(8)(9) and was quickly adapted as a technique for in vivo tumor induction and validation of cancer genes (10)(11)(12)(13)(14)(15). By transplanting CRISPR librarytransduced cancer cells into immunocompromised mice, several genes involved in growth and metastasis of human lung cancer were identified (16).…”

“…These screens typically start with an immunocomprised genetic background or a genetic background carrying multiple pre-engineered mutations, and thus the results may not be applicable to wild-type mice (1,5). They usually need >1 y to obtain tumors (1,3,15). Therefore, there is a strong need for an alternative delivery system that can overcome these shortcomings and be used for direct in vivo CRISPR screening.…”

“…In addition to lentivirus/ retrovirus, the only good choices to mediate efficient genomic integrations are transposons. Both piggyBac (PB) and Sleeping Beauty (SB) transposons have been used for the delivery of individual gRNAs (15,17), suggesting they might be adapted to deliver and express a genome-wide single guide RNA (sgRNA) library for high-throughput screening.…”

CRISPR genome editing and its medical applications

“…Today, the CRISPR system has wide potential for applications in the field of cancer, including epigenetic cancer therapy [4], turning the genes involved in cancer development on and off, modelling cancer, examining the protein domains involved in cancer to evaluate the drug targets, reviewing and modelling the cancers in which several genes are involved or cancers that are caused by chromosomal clutter and rearrangements, and identifying the genes involved in cancer development [3,[43][44][45]. According to various genetic and epigenetic factors in cancer development, cancer modelling using the CRISPR system is expected play an important role in the detection and the identification of factors involved in cancer.…”