CRISPR screening approaches in breast cancer research

Abstract

The emergence of CRISPR-Cas9 technology has transformed functional genomics, offering unmatched opportunities to dissect and understand biological pathways and identify novel therapeutic targets in cancer. Breast cancer is a complex, heterogeneous disease and remains a major cause of morbidity and mortality in women, particularly when diagnosed at advanced or metastatic stages where effective treatments are limited. High-throughput CRISPR screening is undoubtedly a powerful tool to discover novel drug targets, uncover synthetic lethal interactions, and identify vulnerabilities in cancer. This review focuses on advances in our understanding of breast cancer developed through CRISPR-based screening technology, particularly in identifying drivers of breast cancer progression, growth, and metastasis, as well as in identifying potential new therapeutic targets and combination therapies. We discuss recent discoveries, current challenges, and limitations of this approach and explore how advancements in CRISPR technology could have a profound impact on the future of breast cancer treatment.

Keywords: Breast cancer; CRISPR; Drug discovery; Drug resistance; Functional genomics; Novel therapies; Screening.

Fig. 1

Overview of CRISPR-Cas systems and mechanisms of action. Diagram showing the different CRISPR-based systems used in functional genomics screens. The CRISPR-Cas9 system uses sgRNAs to direct the Cas9 endonuclease to target genes where it induces DSBs in the DNA, resulting in gene knockout through error-prone DNA repair mechanisms. CRISPRa uses a catalytically inactive Cas9 (dCas9) fused to a transcriptional activator. sgRNAs are designed to target upstream of the promoter region or translation start site of the target gene, inducing upregulation of the target. CRISPRi works similarly; however, it utilises a transcriptional repressor to silence target genes. Base editing through Cas9-conjugated deaminase can convert bases, inducing substitution mutations in DNA or RNA. Epigenetic modulation is also possible, where dCas9 is conjugated to an epigenetic effector that induces histone modifications or DNA methylation at the promoter of target genes, resulting in chromatin changes, altering gene transcription. Cas13 is an RNA-targeting enzyme that cleaves RNA molecules such as mRNA and lncRNA to silence gene expression without binding to DNA or inducing DNA damage

Fig. 2

Workflow of a pooled CRISPR-Cas9 screening experiment. Schematic representation of the experimental workflow of a pooled CRISPR-Cas9 screen. Initially, a genome-wide or targeted sgRNA library is amplified and packaged into lentiviral particles through transfection of HEK293T cells with the library and packaging plasmids. The lentivirus is harvested, and the viral titre is determined. Cas-expressing cells are then infected with the lentiviral library at a low multiplicity of infection (MOI), increasing the probability that each cell is transduced with a single sgRNA. The resulting heterogeneous pool of cells is then exposed to a selection pressure, either in vivo or in vitro, to enrich or deplete specific sgRNAs based on their impact on cell phenotype. Genomic DNA is then extracted from the final and initial cell populations, and sgRNAs are amplified by PCR then subjected to next-generation sequencing. This quantifies the abundance of each sgRNA, enabling the identification of genes associated with resistance to drugs, growth, metastasis, or other phenotypic responses

Fig. 3

Applications of CRISPR-Cas9 screening experiments. Schematic representation of the different experimental procedures commonly used in breast cancer research. The figure shows how pooled CRISPR screens can undergo selection in different systems to identify drivers of oncogenic processes, such as metastasis, growth, and sensitivity to therapies, and in order to find synthetic lethal gene or drug interactions