Anti-CRISPRs: Unveiling Mechanisms, Discovery, and Applications in the Ongoing Arms Race (2020-2024)
This mini-review examines recent anti-CRISPR research, focusing on proteins that inhibit CRISPR-Cas systems. It covers discovery methods, inhibition mechanisms, and applications in genome editing, highlighting the evolutionary arms race between prokaryotic immunity and mobile genetic elements.
Introduction
The CRISPR-Cas system provides adaptive immunity to prokaryotes against invading mobile genetic elements (MGEs) such as viruses and plasmids. In response, MGEs have evolved anti-CRISPR (Acr) proteins to inhibit CRISPR-Cas systems, leading to an ongoing evolutionary arms race. This mini-review summarizes recent advancements in the field of anti-CRISPR research over the past five years, focusing on the discovery, mechanistic understanding, and applications of Acrs.
Discovery and Characterization of Novel Anti-CRISPRs
A significant focus has been on identifying and characterizing novel Acrs. Joseph Bondy-Denomy's lab has been instrumental in the discovery of multiple Acrs, highlighting the clustering of anti-defense genes in MGEs (Rafael Pinilla‐Redondo et al., 2020, Nature Communications). Their work also demonstrated that broad-spectrum Acrs facilitate horizontal gene transfer (Caroline Mahendra et al., 2020, Nature Microbiology) and explored mobile element warfare via CRISPR and anti-CRISPR in Pseudomonas aeruginosa (Lina M. León et al., 2020, Nucleic Acids Research; Lina M. León et al., 2021, Nucleic Acids Research). Rotem Sorek's group has also contributed to the understanding of how phages overcome bacterial immunity via diverse anti-defence proteins (Erez Yirmiya et al., 2022, Nature; Erez Yirmiya et al., 2023, Nature).
Computational approaches have also accelerated Acr discovery. Jennifer A. Doudna's and Fayyaz Minhas's groups demonstrated the use of machine learning to predict new Acr proteins (Simon Eitzinger et al., 2020, Nucleic Acids Research). Trevor Lithgow's laboratory developed PaCRISPR, a server for predicting and visualizing Acr proteins (Jiawei Wang et al., 2020, Nucleic Acids Research), and AcrHub, an integrative hub for investigating, predicting, and mapping Acr proteins (Jiawei Wang et al., 2020, Nucleic Acids Research). Yanbin Yin's group created AcrFinder, a tool for genome mining Acr operons in prokaryotes and their viruses (Haidong Yi et al., 2020, Nucleic Acids Research). Jiangning Song's group developed PreAcrs, a machine learning framework for identifying anti-CRISPR proteins (Lin Zhu et al., 2022, BMC Bioinformatics). Kira S. Makarova's group reviewed in silico approaches for prediction of anti-CRISPR proteins (Kira S. Makarova et al., 2023, Journal of Molecular Biology). Eugene V. Koonin's group searched for origins of anti-CRISPR proteins by structure comparison (Harutyun Sahakyan et al., 2023, The CRISPR Journal). Akintunde Emiola's group used structure-guided discovery of anti-CRISPR and anti-phage defense proteins (Ning Duan et al., 2023, Nature Communications; Ning Duan et al., 2024, Nature Communications).
Structural and Mechanistic Insights into Anti-CRISPR Function
Understanding the mechanisms by which Acrs inhibit CRISPR-Cas systems is crucial. Blake Wiedenheft's group provided a comprehensive review of the structures and strategies of Acr-mediated immune suppression (Tanner Wiegand et al., 2020, Annual Review of Microbiology). They also showed that AcrIF9 tethers non-sequence specific dsDNA to the CRISPR RNA-guided surveillance complex (Marscha Hirschi et al., 2020, Nature Communications). Alan R. Davidson's lab demonstrated that AcrIF9 functions by inducing the CRISPR-Cas complex to bind DNA non-specifically (Wangting Lu et al., 2020, Nucleic Acids Research; Wangting Lu et al., 2021, Nucleic Acids Research).
Several studies have focused on the structural basis of Acr function. Zhiwei Huang's and Wah Chiu's groups used cryo-EM to reveal the inhibition mechanisms of AcrF9, AcrF8, and AcrF6 against the type I-F CRISPR-Cas complex (Kaiming Zhang et al., 2020, Proceedings of the National Academy of Sciences). Leifu Chang's group determined the structural basis for inhibition of the type I-F CRISPR-Cas surveillance complex by AcrIF4, AcrIF7, and AcrIF14 (C. Gabel et al., 2020, Nucleic Acids Research). Yue Feng's lab provided insights into the inhibition of the type I-F CRISPR-Cas system by the multifunctional AcrIF24 (Lingguang Yang et al., 2021, Nature Communications; Lingguang Yang et al., 2022, Nature Communications), showed that AcrIF5 specifically targets the DNA-bound CRISPR-Cas surveillance complex for inhibition (Yongchao Xie et al., 2021, Nature Chemical Biology; Yongchao Xie et al., 2022, Nature Chemical Biology), and elucidated the dual functions of AcrIF14 during the inhibition of the type I-F CRISPR-Cas surveillance complex (Xi Liu et al., 2021, Nucleic Acids Research). Hongnan Liu's group determined the structural basis of Staphylococcus aureus Cas9 inhibition by AcrIIA14 (Hongnan Liu et al., 2021, Nucleic Acids Research). Hang Yin's and Heng Zhang's groups investigated the inhibition mechanisms of CRISPR-Cas9 by AcrIIA17 and AcrIIA18 (Xiaoshen Wang et al., 2021, Nucleic Acids Research). Wei Sun's and Yanli Wang's groups showed that AcrIIC5 is a dsDNA mimic that inhibits type II-C Cas9 effectors by blocking PAM recognition (Wei Sun et al., 2021, Nucleic Acids Research; Wei Sun et al., 2022, Nucleic Acids Research; Wei Sun et al., 2023, Nucleic Acids Research). Euiyoung Bae's and Jeong‐Yong Suh's groups determined the structure of AcrIE4-F7, revealing a common strategy for dual CRISPR inhibition by targeting PAM recognition sites (Sung-Hyun Hong et al., 2022, Nucleic Acids Research). Hyun Ho Park's group elucidated the molecular basis of dual anti-CRISPR and auto-regulatory functions of AcrIF24 (Gi Eob Kim et al., 2022, Nucleic Acids Research) and the molecular basis of anti-CRISPR operon repression by Aca10 (So Yeon Lee et al., 2022, Nucleic Acids Research).
Regulation and Applications of Anti-CRISPRs
The regulation of Acr expression and their potential applications are also areas of active research. Chris M. Brown's group demonstrated widespread repression of Acr production by anti-CRISPR-associated proteins (Saadlee Shehreen et al., 2021, Nucleic Acids Research; Saadlee Shehreen et al., 2022, Nucleic Acids Research). Guoxu Song's lab engineered potent and versatile CRISPR-Cas9 inhibitors for chemically controllable genome editing (Guoxu Song et al., 2021, Nucleic Acids Research; Guoxu Song et al., 2022, Nucleic Acids Research) and developed a rapid characterization method for Acr proteins and optogenetically engineered variants using a versatile plasmid interference system (Guoxu Song et al., 2022, Nucleic Acids Research; Guoxu Song et al., 2023, Nucleic Acids Research). Wataru Nomura's group showed that Cas9-Geminin and Cdt1-fused anti-CRISPR protein synergistically increase editing accuracy (Daisuke Matsumoto et al., 2023, FEBS Letters) and that SpCas9-HF1 enhances accuracy of cell cycle-dependent genome editing by increasing HDR efficiency, and by reducing off-target effects and indel rates (Daisuke Matsumoto et al., 2024, Molecular Therapy — Nucleic Acids). Ming Li's group showed that CRISPR-repressed toxin–antitoxin provides herd immunity against anti-CRISPR elements (Xian Shu et al., 2024, Nature Chemical Biology). Bingzhi Li's and Xing Zhang's groups showed that Heparin Specifically Inhibits CRISPR/Cas12 Activation, Enabling Ultrasensitive Heparin Detection and Gene Editing Regulation (Min Cao et al., 2024, Analytical Chemistry).
Conclusion
The past five years have witnessed significant progress in the field of anti-CRISPR research. The discovery of novel Acrs, coupled with detailed structural and mechanistic studies, has provided valuable insights into the intricate interplay between CRISPR-Cas systems and their inhibitors. Furthermore, the development of computational tools and the exploration of Acr applications highlight the growing importance of this field in both basic research and biotechnology. Future research should focus on understanding the evolutionary dynamics of Acrs, exploring their potential for regulating CRISPR-Cas systems in diverse applications, and mitigating their impact on genome editing technologies.
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