Applying CRISPR-Cas9 screens to dissect hematological malignancies

Abstract

Bit by bit, over the last few decades, functional genomic tools have been piecing together the molecular puzzle driving tumorigenesis in human patients. Nevertheless, our understanding of the role of several genes and regulatory elements that drive critical cancer-associated physiological processes from disease development to progression to spread is very limited, which significantly affects our ability of applying these insights in the context of improved disease management. The recent advent of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-based technology and its application in cancer genomics has, however, allowed the generation of a wealth of knowledge that has helped decipher several critical questions associated with translational cancer research. Precisely, the high-throughput capability coupled with a high level of technological plasticity associated with the CRISPR-Cas9 screens have expanded our horizons from a mere struggle to appreciate cancer as a genetic disease to observing the integrated genomic/epigenomic network of numerous malignancies and correlating it with our present knowledge of drugging strategies to develop innovative approaches for next-generation precision cancer medicine. Specifically, within blood cancers, current CRISPR screens have specifically focused on improving our understanding of drug resistance mechanisms, disease biology, the development of novel therapeutic approaches, and identifying the molecular mechanisms of current therapies, with an underlying aim of improving disease outcomes. Here, we review the development of the CRISPR-Cas9 genome-editing strategy, explicitly focusing on the recent advances in the CRISPR-Cas9-based screening approaches, its current capabilities, limitations, and future applications in the context of hematological malignancies.

Figure 1.

Overview of the role of genome/gene-editing platforms. Genome-editing nucleases (TALEN, ZFN, and CRISPR-Cas9) induce DSBs within the nuclear DNA at specific sites, which can subsequently be repaired using nonhomologous end-joining (NHEJ) or homology-directed repair (HDR) pathways that assist in targeted modification of the DNA. In contrast, gene-editing RNA interference strategies (using shRNA or small interfering RNA [siRNA]) bind to complementary messenger RNA sequences within the cytoplasm and cause its degradation. mRNA, messenger RNA; RISC, RNA-induced silencing complex. Created with www.BioRender.com.

Figure 2.

Workflow of pooled vs arrayed CRISPR-Cas9 screening approaches. For either screening strategy, the input pool of sgRNAs and Cas9 is designed using complex bioinformatic algorithms to maximize targeting efficiency and limit off-target effects. In the pooled screening approach, a viral sgRNA library generated from the sgRNA/Cas9 complexes can then be delivered to a single vessel of cells at a low MOI. In contrast, within the arrayed screening format viral libraries are delivered to discrete cell populations grown in multiwell plates to allow for unique representation of individual sgRNAs within each well. Following the administration of a challenge (such as drug treatment), based on the selection strategy adopted (positive, negative, or FACS-associated marker based), several rounds of cell selection may be included within pooled screens to allow for targeted cell enrichment. In contrast, the arrayed screens do not require selective enrichment, rather the developing phenotype within the individual cell populations form the raw data that can be visualized in the final step of the assay through high-throughput image acquisition strategies followed by statistical ranking of the observed phenotypes. In contrast, raw data (genomic DNA from the selected cell populations) must be profiled using deep-sequencing approaches followed by an analysis of depleted or enriched sgRNA population to identify the potential genes that could be associated with potential cancer pathways although downstream validation will be required to confirm the speculative role of the potential genes within the disease. Key steps of convergence between the 2 pathways have been highlighted for both pathways. gRNA, guide RNA. Created with www.BioRender.com.