The rise and future of CRISPR-based approaches for high-throughput genomics

The rise and future of CRISPR-based approaches for high-throughput genomics

Abstract

Clustered regularly interspaced short palindromic repeats has revolutionized the field of genome editing. To circumvent the permanent modifications made by traditional CRISPR techniques and facilitate the study of both essential and nonessential genes, CRISPR interference was developed. This gene-silencing technique employs a deactivated Cas effector protein and a guide RNA to block transcription initiation or elongation. Continuous improvements and a better understanding of the mechanism of CRISPRi have expanded its scope, facilitating genome-wide high-throughput screens to investigate the genetic basis of phenotypes. Additionally, emerging CRISPR-based alternatives have further expanded the possibilities for genetic screening. This review delves into the mechanism of CRISPRi, compares it with other high-throughput gene-perturbation techniques, and highlights its superior capacities for studying complex microbial traits. We also explore the evolution of CRISPRi, emphasizing enhancements that have increased its capabilities, including multiplexing, inducibility, titratability, predictable knockdown efficacy, and adaptability to nonmodel microorganisms. Beyond CRISPRi, we discuss CRISPR activation, RNA-targeting CRISPR systems, and single-nucleotide resolution perturbation techniques for their potential in genome-wide high-throughput screens in microorganisms. Collectively, this review gives a comprehensive overview of the general workflow of a genome-wide CRISPRi screen, with an extensive discussion of strengths and weaknesses, future directions, and potential alternatives.

Introduction

Microorganisms have developed complex traits that facilitate adaptation to different environments and interactions with various species. A microbial trait can be defined as a phenotypic characteristic, including physiology, morphology, or behaviour, and is determined by genetic loci. Often, traits are controlled by multiple genetic loci, resulting in complex genetic interaction networks. Moreover, environmental conditions and interactions with other species can modify traits, contributing to increased complexity. Such trait modifications can occur when microorganisms modify their DNA or alter their expression levels through post-transcriptional or post-translational changes. These effects can radically alter phenotypes, for example by adapting the growth rate, shifting metabolic processes to match changing environmental conditions, or by providing resistance against certain stressors. Nevertheless, the transient nature of some phenotypes can render certain traits even more complex to study. A well-known transient phenotype is tolerance, which can emerge through phenotypic switching in the absence of genetic changes. For example, microorganisms can shield themselves against unfavourable conditions by temporarily adopting a metabolically inactive state, resulting in the formation of spores or persister cells. To investigate the entry and exit from this metabolically inactive state, it is important to consider temporal regulation of gene expression. Furthermore, environmental factors are also important. For example, tolerance is influenced by nutrient availability, as exemplified by triggered sporulation upon entering stationary phase or by the increased presence of persister cells during stationary phase compared to exponential phase. Additionally, species interactions influence traits. For example, tolerance of Staphylococcus aureus to antibiotics is increased in the presence of Pseudomonas aeruginosa. In short, microbial traits can be time-, environment-, and interaction-dependent, rendering the study of underlying genetics inherently complex.

Although advances in several high-throughput screening methods have tremendously facilitated the investigation of genotype-phenotype relationships, deciphering the genetics behind complex microbial traits remains challenging. Important impediments in many high-throughput genetic screening methods include: (i) the lack of a reversible system, (ii) the limitation to nonessential genes, (iii) the labour-intensive nature of developing genome-wide libraries, and (iv) the challenge of constructing manageable-sized libraries to study phenotypes under bottleneck conditions, for example in in vivo studies. This shows the need for an optimized high-throughput genome-wide screening platform coping with these limitations.

A remarkable evolution in recent years has been the development and implementation of clustered regularly interspaced short palindromic repeats-based approaches, with CRISPR interference emerging as an important gene-silencing technique. This innovative technique addresses many of the previously mentioned challenges in high-throughput genetic screening approaches, thereby unlocking new opportunities for exploring complex microbial traits. In this review, we will compare CRISPRi to currently used high-throughput approaches and illustrate why it stands out as a promising tool to study complex microbial phenotypes. We will provide a comprehensive overview of its mechanism and use for genome-wide screening, discuss important limitations and how they can be overcome, and review promising improvements that have enhanced its capabilities. Likewise, we discuss other CRISPR-based approaches that are currently being developed or optimized and that may overcome persisting limitations in genome-wide high-throughput screening or offer complementary strategies for studying the genetic basis of phenotypes.