CRISPR Screens in Synthetic Lethality and Combinatorial Therapies for Cancer

Abstract

Cancer is a complex disease resulting from the accumulation of genetic dysfunctions. Tumor heterogeneity causes the molecular variety that divergently controls responses to chemotherapy, leading to the recurrent problem of cancer reappearance. For many decades, efforts have focused on identifying essential tumoral genes and cancer driver mutations. More recently, prompted by the clinical success of the synthetic lethality (SL)-based therapy of the PARP inhibitors in homologous recombinant deficient tumors, scientists have centered their novel research on SL interactions (SLI). The state of the art to find new genetic interactions are currently large-scale forward genetic CRISPR screens. CRISPR technology has rapidly evolved to be a common tool in the vast majority of laboratories, as tools to implement CRISPR screen protocols are available to all researchers. Taking advantage of SLI, combinatorial therapies have become the ultimate model to treat cancer with lower toxicity, and therefore better efficiency. This review explores the CRISPR screen methodology, integrates the up-to-date published findings on CRISPR screens in the cancer field and proposes future directions to uncover cancer regulation and individual responses to chemotherapy.

Keywords: CRISPR screen; cancer therapeutic resistance; combinatorial therapy; synthetic lethality.

Figure 1

Schematic outline of CRISPR/Cas genome-wide screens. (A) Previous to the screen, the experimental design must fit the biological question to solve. The experimental strategy includes selection of cell line/s, CRISPR library and its representation, number of biological and technical replicates and the total amount of samples to harvest, all together will dictate cell quantity and number of plates to culture. (B) Before starting the actual experiment, a setup preparation takes place: characterization of the cell line/s, integration of the endonuclease Cas9, library amplification, viral particles preparation and cell transduction. (C) The screening steps are: selection of the transduced cells, collection of the basal samples, drug treatment or pressure selection, samples harvest, DNA extraction, PCR amplification, NGS, computational analysis and candidate compilation. (D) For candidate hits validation, gene expression may be repressed by RNAi or shRNA or genes may be deleted using CRISPR KO, in vitro and in vivo.

Figure 2

(A) Proof of principle CRISPR/Cas screen. 1. Minilibrary design with 3 sgRNAs for each gene of interest (3 genes that when depleted give PARPi resistance in BRCA2 background) plus 3 NT sgRNAs. 2. sgRNAs choice (at 5′ common exons, with minimal off-targets). 3. sgRNAs cloning into LentiGuide-Puro vector. 4. Minilibrary constructs amplification and lentiviral particles production. 5. Low MOI transduction into BRCA2 fibroblast cells previously transduced with the plasmid LentiCas9-Blast. 6. Puromycin selection of cells with transduced sgRNAs, during 9 days. 7. Screen: non-treated and treated samples collection, after 7 days of 100 nM olaparib. 8. PCR of the sgRNA region. 9. NGS by MiSeq. 10. FASTQ files analysis and mapping to sgRNA library. 11. Statistic significant candidate hits by quantification of the relative proportion of every sgRNA before and after the selection. (B) Relative percentage of sgRNAs in olaparib treated samples versus non-treated. Average of the three-targeted genes and the NT sgRNAs. Only sgRNAs with significant p-values from the student t-test statistics are shown. Values: CHD4 118.4%, 2 sgRNAs; MRE11A 102.6%, 3 sgRNAs, PTIP 103.5%, 1 sgRNA and NT 94.5% data from four sgRNA with no significant p-values (CHD4_3, PTIP_1, PTIP_3 and NT_2).