Functional Genomics via CRISPR–Cas

Abstract

RNA-guided CRISPR (clustered regularly interspaced short palindromic repeat)-associated Cas proteins have recently emerged as versatile tools to investigate and engineer the genome. The programmability of CRISPR-Cas has proven especially useful for probing genomic function in high-throughput. Facile single-guide RNA library synthesis allows CRISPR-Cas screening to rapidly investigate the functional consequences of genomic, transcriptomic, and epigenomic perturbations. Furthermore, by combining CRISPR-Cas perturbations with downstream single-cell analyses (flow cytometry, expression profiling, etc.), forward screens can generate robust data sets linking genotypes to complex cellular phenotypes. In the following review, we highlight recent advances in CRISPR-Cas genomic screening while outlining protocols and pitfalls associated with screen implementation. Finally, we describe current challenges limiting the utility of CRISPR-Cas screening as well as future research needed to resolve these impediments. As CRISPR-Cas technologies develop, so too will their clinical applications. Looking ahead, patient centric functional screening in primary cells will likely play a greater role in disease management and therapeutic development.

Research Techniques Made Simple: CRISPR Genetic Screens

“…CRISPR-based screens demonstrate improved versatility, efficacy, and lower off-target effects compared with approaches such as RNAi (Ford et al, 2019;Guitart et al, 2016;Schuster et al, 2019). For a comprehensive description of the fundamentals of CRISPR-mediated genome editing, we refer to a previous Research Techniques Made Simple article (Guitart et al, 2016).…”

“…CRISPR screens can be performed in primary cells, such as keratinocytes, melanocytes, and fibroblasts (Fenini et al, 2018;Slivka et al, 2019;Sun et al, 2015). However, primary cells can be difficult to transfect, generate lower geneediting efficiency, or may have cell limitations that are incompatible with long-term library screens (Ford et al, 2019). For these reasons, use of transformed or immortalized cell lines, such as 293T or HeLa cells, are sometimes favored for their technical tractability.…”

“…CRISPR-based genome editing requires two components, the Cas9 protein and the sgRNA, which contains both a scaffold and a target-specific spacer sequence. There are several options to deliver these components into cells, each with their specific advantages and disadvantages ( Table 2 in Ford et al, 2019). Some delivery methods can give rise to undesired effects, such as cytotoxicity or innate immunity responses (Kim et al, 2018).…”

“…The human genome contains over 20,000 protein-coding genes, and their disruption underlies many diseases, underscoring the need to comprehensively understand their biological functions. One approach to studying gene functions and biological phenotypes is to perform a genetic screen, which aims for systematic functional interrogation of many candidate elements in a single experiment (Doench, 2018;Ford et al, 2019;Sanjana, 2017;Schuster et al, 2019). In a typical cell cultureebased screen, systematic loss-of-function of a set of candidates is applied to identify elements contributing to a phenotype of interest.…”

“…In a typical cell cultureebased screen, systematic loss-of-function of a set of candidates is applied to identify elements contributing to a phenotype of interest. RNA interference (RNAi) and transposon-based technologies have been used successfully, but since their development, CRISPR/Cas-based tools have become a preferred method for genetic screens (Doench, 2018;Ford et al, 2019;Guitart et al, 2016;Schuster et al, 2019).…”