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The escape of CRISPR-mediated gene editing in Zymomonas mobilis.
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems have been widely applied for gene or genome editing. Adequate checking is important to screen mutants after CRISPR-mediated editing events. Here, we report gene escape cases after the knockout by Type I-F native CRISPR system in Zymomonas mobilis. Through amplifying both the gene of interest and its flanking homologous arms, followed by curing the editing plasmid, we found different destinies for gene-editing events. Some genes were readily knocked out and followed by the easy plasmid curing. In some other cases, however, the editing plasmid was difficult to remove from the cell, or the deleted genes were transferred into the editing plasmid. For example, the targeted region of fur can be integrated into the editing plasmid after the knockout, resulting in a spurious editing event. We supposed that the transfer of the gene may be attributed to bacterial insertion sequences. Searching for literatures on the gene knockout using CRISPR in bacteria reveals that the escape event is likely underestimated due to inadequate validation in other microbes. Hence, several strategies are proposed to enhance gene knockout and plasmid curing.
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CRISPR editing within microbial communities.
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Powerful Microbial Base-Editing Toolbox: From Optimization Strategies to Versatile Applications.
Base editors (BE) based on CRISPR systems are practical gene-editing tools which continue to drive frontier advances of life sciences. BEs are able to efficiently induce point mutations at target sites without double-stranded DNA cleavage. Hence, they are widely employed in the fields of microbial genome engineering. As applications of BEs continue to expand, the demands for base-editing efficiency, fidelity, and versatility are also on the rise. In recent years, a series of optimization strategies for BEs have been developed. By engineering the core components of BEs or adopting different assembly methods, the performance of BEs has been well optimized. Moreover, series of newly established BEs have significantly expanded the base-editing toolsets. In this Review, we will summarize the current efforts for BE optimization, introduce several novel BEs with versatility, and look forward to the broadened applications for industrial microorganisms.
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Thinking About CRISPR: The Ethics of Human Genome Editing.
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CRISPRoffT: comprehensive database of CRISPR/Cas off-targets.
The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated protein) programmable nuclease system continues to evolve, with in vivo therapeutic gene editing increasingly applied in clinical settings. However, off-target effects remain a significant challenge, hindering its broader clinical application. To enhance the development of gene-editing therapies and the accuracy of prediction algorithms, we developed CRISPRoffT (https://ccsm.uth.edu/CRISPRoffT/). Users can access a comprehensive repository of off-target regions predicted and validated by a diverse range of technologies across various cell lines, Cas enzyme variants, engineered sgRNAs (single guide RNAs) and CRISPR editing systems. CRISPRoffT integrates results of off-target analysis from 74 studies, encompassing 29 experimental prediction techniques, 368 guide sequences, 226 164 potential guide and off-target pairs and 8840 validated off-targets. CRISPRoffT features off-target data from different CRISPR approaches (knockout, base editing and prime editing) applied under diverse experimental conditions, including 85 different Cas/guide RNA (gRNA) combinations used across 34 different human and mouse cell lines. CRISPRoffT provides results of comparative analyses for individual guide sequences, genes, cell types, techniques and Cas/gRNA combinations under different conditions. CRISPRoffT is a unique resource providing valuable insights that facilitate the safety-driven design of CRISPR-based therapeutics, inform experimental design, advance the development of computational off-target prediction algorithms and guide RNA design algorithms.
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[Research progress of CRISPR-Cas9 system for gene therapy].
The clustered regulatory interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) system is the part of the prokaryotic immune system, which could recognize and delete the exogenous sequences originated from virus or plasmid. Based on its mechanism, CRISPR-Cas9 system was developed into the new generation of gene editing tool. Compared to the existed technologies such as ES targeting, ZFN or TALEN, CRISPR-Cas9 system is a more efficient, economical and promising approach to manipulate the genome. In this review, we summarize the research progress about CRISPR-Cas9 technology, especially the latest applications in gene therapy studies of human diseases.
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Development of CRISPR/Cas9 for Efficient Genome Editing in Toxoplasma gondii.
Efficient and site-specific alteration of the genome is key to decoding and altering the genomic information of an organism. Over the last couple of years, the RNA-guided Cas9 nucleases derived from the prokaryotic type 2 CRISPR (clustered regularly interspaced short palindromic repeats) systems have drastically improved our ability to engineer the genomes of a variety of organisms including Toxoplasma gondii. In this chapter, we describe detailed protocols for using the CRISPR/Cas9 system adapted from Streptococcus pyogenes to perform efficient genetic manipulations in T. gondii such as gene disruption, gene tagging and genetic complementation. The technical details of the strategy, including CRISPR plasmid construction, target construct generation, parasite transfection and positive clone identification are also provided. These methods are easy to customize to any gene of interest (GOI) and will greatly accelerate studies on this important pathogen.
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Next-generation CRISPR technology for genome, epigenome and mitochondrial editing.
The application of rapidly growing CRISPR toolboxes and methods has great potential to transform biomedical research. Here, we provide a snapshot of up-to-date CRISPR toolboxes, then critically discuss the promises and hurdles associated with CRISPR-based nuclear genome editing, epigenome editing, and mitochondrial editing. The technical challenges and key solutions to realize epigenome editing in vivo, in vivo base editing and prime editing, mitochondrial editing in complex tissues and animals, and CRISPR-associated transposases and integrases in targeted genomic integration of very large DNA payloads are discussed. Lastly, we discuss the latest situation of the CRISPR/Cas9 clinical trials and provide perspectives on CRISPR-based gene therapy. Apart from technical shortcomings, ethical and societal considerations for CRISPR applications in human therapeutics and research are extensively highlighted.
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Recent advances in the CRISPR genome editing tool set.
Genome editing took a dramatic turn with the development of the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated proteins (Cas) system. The CRISPR-Cas system is functionally divided into classes 1 and 2 according to the composition of the effector genes. Class 2 consists of a single effector nuclease, and routine practice of genome editing has been achieved by the development of the Class 2 CRISPR-Cas system, which includes the type II, V, and VI CRISPR-Cas systems. Types II and V can be used for DNA editing, while type VI is employed for RNA editing. CRISPR techniques induce both qualitative and quantitative alterations in gene expression via the double-stranded breakage (DSB) repair pathway, base editing, transposase-dependent DNA integration, and gene regulation using the CRISPR-dCas or type VI CRISPR system. Despite significant technical improvements, technical challenges should be further addressed, including insufficient indel and HDR efficiency, off-target activity, the large size of Cas, PAM restrictions, and immune responses. If sophisticatedly refined, CRISPR technology will harness the process of DNA rewriting, which has potential applications in therapeutics, diagnostics, and biotechnology.
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CRISPR genome editing using a combined positive and negative selection system.
The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system is a powerful genome editing tool that has revolutionized research. Single nucleotide polymorphisms (SNPs) are the most common form of genetic variation in humans. Only a subset of these SNPs has been shown to be linked to genetic diseases, while the biological relevance of the majority remains unclear. Investigating these variants of unknown significance could provide valuable insights into their roles in biological processes, disease susceptibility, and treatment responses. While CRISPR/Cas has emerged as a transformative technology, its ability to make single nucleotide substitutions remains a significant limitation. Other techniques in single nucleotide editing, such as base editing and prime editing, offer promising possibilities to complement CRISPR/Cas systems, though they also have their own limitations. Hence, alternative approaches are necessary to overcome the limitations of CRISPR. Here, to improve the feasibility of generating single base edits in the genome, we provide a protocol that introduces a multiple expression and dual selection (MEDS) system, which, alongside CRISPR, utilizes the opposing roles of cytosine deaminase/uracil phosphoribosyltransferase (CD/UPRT) for negative selection and neomycin phosphotransferase II (NPT II) for positive selection. As a proof of concept and to demonstrate feasibility of the method, we used MEDS, along with traditional CRISPR-Cas9, to generate sickle hemoglobin by introducing a point mutation (A → T) in the sixth codon of the hemoglobin beta gene.
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