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A Generally Applicable CRISPR/Cas9 Screening Technique to Identify Host Genes Required for Virus Infection as Applied to Human Cytomegalovirus (HCMV) Infection of Epithelial Cells.
Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 screens enable virus-host genetic screens to be undertaken in a more robust manner than previously possible and has had a tremendous impact in the field of virus study. Researchers can take advantage of the power of CRISPR genetic screens to discover virus-host interaction genes including host receptors and signaling molecules (Bazzone et al., mBio 10 (1): e02734-18, 2019; E et al., Proc Natl Acad Sci U S A 116(14):7043-7052, 2019; McDougall et al., Curr Opin Virol 29:87-100, 2018; Savidis et al., Cell Rep 16(1):232-246, 2016). In principle, lysis of cells late in the virus infection cycle allows one to screen for essential genes using pooled single-guide RNAs (sgRNAs) that collective target an entire host cell genome simply by identifying mutant cells that are resistant to virus-induced cell death. Here we focus on using this technique on epithelial cells to identify host targets required for human cytomegalovirus (HCMV) infection.
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Genome editing: the road of CRISPR/Cas9 from bench to clinic.
Molecular scissors engineered for site-specific modification of the genome hold great promise for effective functional analyses of genes, genomes and epigenomes and could improve our understanding of the molecular underpinnings of disease states and facilitate novel therapeutic applications. Several platforms for molecular scissors that enable targeted genome engineering have been developed, including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and, most recently, clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated-9 (Cas9). The CRISPR/Cas9 system's simplicity, facile engineering and amenability to multiplexing make it the system of choice for many applications. CRISPR/Cas9 has been used to generate disease models to study genetic diseases. Improvements are urgently needed for various aspects of the CRISPR/Cas9 system, including the system's precision, delivery and control over the outcome of the repair process. Here, we discuss the current status of genome engineering and its implications for the future of biological research and gene therapy.
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Human Germline Genome Editing.
With CRISPR/Cas9 and other genome-editing technologies, successful somatic and germline genome editing are becoming feasible. To respond, an American Society of Human Genetics (ASHG) workgroup developed this position statement, which was approved by the ASHG Board in March 2017. The workgroup included representatives from the UK Association of Genetic Nurses and Counsellors, Canadian Association of Genetic Counsellors, International Genetic Epidemiology Society, and US National Society of Genetic Counselors. These groups, as well as the American Society for Reproductive Medicine, Asia Pacific Society of Human Genetics, British Society for Genetic Medicine, Human Genetics Society of Australasia, Professional Society of Genetic Counselors in Asia, and Southern African Society for Human Genetics, endorsed the final statement. The statement includes the following positions. (1) At this time, given the nature and number of unanswered scientific, ethical, and policy questions, it is inappropriate to perform germline gene editing that culminates in human pregnancy. (2) Currently, there is no reason to prohibit in vitro germline genome editing on human embryos and gametes, with appropriate oversight and consent from donors, to facilitate research on the possible future clinical applications of gene editing. There should be no prohibition on making public funds available to support this research. (3) Future clinical application of human germline genome editing should not proceed unless, at a minimum, there is (a) a compelling medical rationale, (b) an evidence base that supports its clinical use, (c) an ethical justification, and (d) a transparent public process to solicit and incorporate stakeholder input.
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Zinc Fingers, TALEs, and CRISPR Systems: A Comparison of Tools for Epigenome Editing.
The completion of genome, epigenome, and transcriptome mapping in multiple cell types has created a demand for precision biomolecular tools that allow researchers to functionally manipulate DNA, reconfigure chromatin structure, and ultimately reshape gene expression patterns. Epigenetic editing tools provide the ability to interrogate the relationship between epigenetic modifications and gene expression. Importantly, this information can be exploited to reprogram cell fate for both basic research and therapeutic applications. Three different molecular platforms for epigenetic editing have been developed: zinc finger proteins (ZFs), transcription activator-like effectors (TALEs), and the system of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) proteins. These platforms serve as custom DNA-binding domains (DBDs), which are fused to epigenetic modifying domains to manipulate epigenetic marks at specific sites in the genome. The addition and/or removal of epigenetic modifications reconfigures local chromatin structure, with the potential to provoke long-lasting changes in gene transcription. Here we summarize the molecular structure and mechanism of action of ZF, TALE, and CRISPR platforms and describe their applications for the locus-specific manipulation of the epigenome. The advantages and disadvantages of each platform will be discussed with regard to genomic specificity, potency in regulating gene expression, and reprogramming cell phenotypes, as well as ease of design, construction, and delivery. Finally, we outline potential applications for these tools in molecular biology and biomedicine and identify possible barriers to their future clinical implementation.
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Engineering DNA-Targeting CRISPR and CRISPR-Like Effectors: Advances from Rational Design and High-Throughput Screening to AI-Driven Development.
Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins constitute adaptive immune systems in prokaryotes and have transformed life sciences, precision medicine, and synthetic biology as programmable genome-editing tools. Despite their broad utility, naturally occurring DNA-targeting Cas effectors remain constrained by several intrinsic limitations, including large protein size that complicates delivery, stringent protospacer adjacent motif (PAM) requirements that restrict targetable genomic space, and mismatch tolerance that can lead to off-target activity and potential genotoxicity. These challenges have made Cas protein engineering and the discovery of novel CRISPR and CRISPR-like systems from metagenomic resources central to the development of next-generation genome-editing platforms. This Review places recent advances within an integrated synthetic biology engineering continuum that links natural effector discovery, structure-guided hypothesis generation, high-throughput functional screening, machine learning-enabled model construction, and iterative redesign. This Review summarizes progress in the screening, optimization, and functional engineering of DNA-targeting CRISPR and CRISPR-like effectors, with emphasis on structure-guided rational design, directed evolution coupled with high-throughput screening, bioinformatics- and evolution-guided mining of novel systems from large-scale sequence databases, and artificial intelligence-assisted development. By integrating these strategies, we highlight how CRISPR effector engineering is moving toward design-build-test-learn (DBTL)-inspired workflows that expand the functional landscape of genome-editing technologies and advance genome editing toward improved efficiency, safety, and programmability.
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Virus-Based CRISPR/Cas9 Genome Editing in Plants.
CRISPR/Cas9 is a versatile tool for plant gene function studies and crop improvement. However, traditional CRISPR/Cas9-mediated genome editing requires plant tissue culture that is both time-consuming and genotype-dependent. Ma et al. recently reported a novel virus-based method for delivering CRISPR/Cas9 into plant cells, and this should further expand the application of CRISPR/Cas9-mediated genome editing.
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Epigenome editing by CRISPR/Cas9 in clinical settings: possibilities and challenges.
Epigenome editing is a promising approach for both basic research and clinical application. With the convergence of techniques from different fields, regulating gene expression artificially becomes possible. From a clinical point of view, targeted epigenome editing by CRISPR/Cas9 of disease-related genes offers novel therapeutic avenues for many diseases. In this review, we summarize the EpiEffectors used in epigenome editing by CRISPR/Cas9, current applications of epigenome editing and progress made in this field. Moreover, application challenges such as off-target effects, inefficient delivery, stability and immunogenicity are discussed. In conclusion, epigenome editing by CRISPR/Cas9 has broad prospects in the clinic, and future work will promote the application of this technology.
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Finally, Archaea Get Their CRISPR-Cas Toolbox.
The majority of archaea encode CRISPR-Cas systems but only a few CRISPR-Cas-based genetic tools have been developed for organisms from this domain. Nayak and Metcalf have harnessed a bacterial Cas9 protein for genome editing in Methanosarcina acetivorans, enabling efficient gene deletion and replacement.
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Harnessing Deep Learning Models for Guide RNA Optimization and Off-Target Prediction in CRISPR Systems.
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-based genome and transcriptome editing technologies have emerged as powerful tools for therapeutic, agricultural, and industrial applications. However, their broader clinical and translational use remains limited by variable guide RNA (gRNA) or single-guide RNA (sgRNA) efficiency and unintended off-target activity, which may lead to genotoxic effects and major safety concerns. To address these challenges, recent research has increasingly shifted from heuristic scoring approaches and traditional machine learning (ML) methods toward deep learning (DL) models capable of learning complex sequence-function relationships from large-scale experimental datasets generated by assays such as GUIDE-seq (Genome-wide Unbiased Identification of Double-stranded Breaks Enabled by Sequencing), CIRCLE-seq (Circularization for In Vitro Reporting of Cleavage Effects by Sequencing), and CHANGE-seq (Cumulative and Homology-independent Analysis of Nuclease Genome-wide Effects by Sequencing). This review critically examines recent advances in DL approaches for gRNA optimization and off-target prediction in CRISPR systems. We discuss the development of convolutional neural networks (CNNs), recurrent neural networks (RNNs), transformer-based architectures, and foundation models designed to improve prediction accuracy, specificity, and generalizability across diverse biological contexts.
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Engineered extracellular vesicles as versatile ribonucleoprotein delivery vehicles for efficient and safe CRISPR genome editing.
Transient delivery of CRISPR-based genome editing effectors is important to reduce off-target effects and immune responses. Recently extracellular vesicles (EVs) have been explored for Cas9 ribonucleoprotein (RNP) delivery. However, lack of mechanisms to enrich RNPs into EVs limited the efficiency of EVs as a RNP delivery vehicle. Here we describe a mechanism to actively enrich RNPs into EVs. We used the specific interaction between RNA aptamer and aptamer-binding protein (ABP) to enrich RNPs into EVs. We inserted RNA aptamer com into single guide RNA (sgRNA), and fused com-binding ABP Com to both termini of tetraspan protein CD63 that is abundant in exosomes. We found that the Com/com interaction enriched Cas9 and adenine base editor (ABE) RNPs into EVs, via forming a three-component complex including CD63-Com fusion protein, com-modified sgRNA and Cas9 or ABE. The RNP enriched EVs are efficient in genome editing and transiently expressed. The system is capable of delivering RNPs targeting multiple loci for multiplex genome editing. In addition, Cas9 from different species can be used together. The EV-delivered RNPs are active in vivo. The data show that the aptamer and ABP interactions can be utilized to actively enrich RNPs into EVs for improved genome editing efficiency and safety.
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