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Multiplex CRISPR-Cas Genome Editing: Next-Generation Microbial Strain Engineering.
Genome editing is a crucial technology for obtaining desired phenotypes in a variety of species, ranging from microbes to plants, animals, and humans. With the advent of CRISPR-Cas technology, it has become possible to edit the intended sequence by modifying the target recognition sequence in guide RNA (gRNA). By expressing multiple gRNAs simultaneously, it is possible to edit multiple targets at the same time, allowing for the simultaneous introduction of various functions into the cell. This can significantly reduce the time and cost of obtaining engineered microbial strains for specific traits. In this review, we investigate the resolution of multiplex genome editing and its application in engineering microorganisms, including bacteria and yeast. Furthermore, we examine how recent advancements in artificial intelligence technology could assist in microbial genome editing and engineering. Based on these insights, we present our perspectives on the future evolution and potential impact of multiplex genome editing technologies in the agriculture and food industry.
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Establishment of an efficient Agrobacterium-mediated transformation system and CRISPR/Cas9-mediated genome editing of the OvPDS1 gene in Oxalis vulcanicola 'Sunset Velvet'.
This study overcomes a key technical barrier by establishing transformation and enabling first CRISPR/Cas9 editing in Oxalis, providing a platform for functional genomics and breeding. The lack of an efficient genetic transformation system has considerably hindered functional genomics studies in Oxalis vulcanicola 'Sunset Velvet'. Here, we established a stable and efficient Agrobacterium-mediated transformation system using stem segments as explants. Key parameters, including pre-culture duration, infection time, Agrobacterium cell density, acetosyringone (AS) concentration, and co-cultivation period, were systematically optimized. Under optimal conditions, the highest transient β-glucuronidase (GUS) expression rate reached approximately 9.0%, and eight stable transgenic lines were successfully obtained. CRISPR/Cas9-mediated genome editing was achieved in Oxalis for the first time. Targeted mutagenesis of OvPDS1, a gene involved in carotenoid biosynthesis, resulted in an albino phenotype, and Sanger sequencing confirmed a base substitution at the target site. Although the editing efficiency was relatively low (0.5%), this result demonstrates the feasibility of genome editing in Oxalis. This study overcomes a major technical bottleneck and provides a robust platform for functional gene analysis, trait improvement, and molecular breeding in O. vulcanicola 'Sunset Velvet' and other non-model ornamental plants.
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Single-Nucleotide Microbial Genome Editing Using CRISPR-Cas12a.
Microbial genome editing can be achieved by donor DNA-directed mutagenesis and CRISPR-Cas12a-mediated negative selection. Single-nucleotide-level genome editing enables the manipulation of microbial cells exactly as designed. Here, we describe single-nucleotide substitutions/indels in the target DNA of E. coli genome using a mutagenic DNA oligonucleotide donor and truncated crRNA/Cas12a system. The maximal truncation of nucleotides at the 3'-end of the crRNA enables Cas12a-mediated single-nucleotide-level precise editing at galK targets in the genome of E. coli.
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Genome editing in rice using CRISPR/Cas12i3.
The CRISPR/Cas type V-I is a family of programmable nuclease systems that prefers a T-rich protospacer adjacent motif (PAM) and is guided by a short crRNA. In this study, the genome-editing application of Cas12i3, a type V-I family endonuclease, was characterized in rice. We developed a CRIPSR/Cas12i3-based Multiplex direct repeats (DR)-spacer Array Genome Editing (iMAGE) system that was efficient in editing various genes in rice. Interestingly, iMAGE produced chromosomal structural variations with a higher frequency than CRISPR/Cas9. In addition, we developed base editors using deactivated Cas12i3 and generated herbicide-resistant rice plants using the base editors. These CRIPSR/Cas12i3-based genome editing systems will facilitate precision molecular breeding in plants.
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Prime Editing: Game Changer for Modifying Plant Genomes.
Prime editing, developed by Anzalone et al., brings genome editing to a new level, because this approach allows introduction of all mutation types, including insertions, deletions, and all putative 12 types of base-to-base conversions. Previously tested in human cells, this technique has been adapted for use in plants by Lin et al.
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Engineered Cas12j-8 is a Versatile Platform for Multiplexed Genome Modulation in Mammalian Cells.
Cas12j-8 is a compact Cas nuclease discovered from the metagenome of giant bacteriophages, consisting of only 717 amino acids and recognizing the '5-TTN-3' protospacer adjacent motif (PAM) sequence. However, its low gene editing efficiency in mammalian cells limits its application in therapeutic gene editing. To address this limitation, structure-guided mutagenesis is employed to replace key negatively charged residues with arginine, strengthening DNA binding. The resulting quintuple mutant, engineered Cas12j-8 (enCas12j-8), demonstrates robust on-target editing efficiency comparable to LbCas12a while maintaining low off-target effects. Cytosine base editors (CBEs) and adenine base editors (ABEs) are developed using enCas12j-8, achieving up to 29.54-fold C-to-T and 36.57-fold A-to-G conversion efficiency compared with the wild-type at the dominated sites, respectively. Notably, enCas12j-8 enables multiplexed editing of three genomic loci simultaneously via a single crRNA array, achieving efficiencies comparable to single-guide approaches. Additionally, enCas12j-8-ABE facilitates the disruption of splice acceptor sites, effectively inducing exon skipping in the SOD1 gene. This strategy holds potential significance for therapeutic genome modulation. These findings establish enCas12j-8 as a versatile, high-precision tool for genome engineering, combining efficient delivery, multiplexing capability, and compatibility with diverse editing modalities.
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Efficient CRISPR/Cas9 genome editing with low off-target effects in zebrafish.
Gene modifications in animal models have been greatly facilitated through the application of targeted genome editing tools. The prokaryotic CRISPR/Cas9 type II genome editing system has recently been applied in cell lines and vertebrates. However, we still have very limited information about the efficiency of mutagenesis, germline transmission rates and off-target effects in genomes of model organisms. We now demonstrate that CRISPR/Cas9 mutagenesis in zebrafish is highly efficient, reaching up to 86.0%, and is heritable. The efficiency of the CRISPR/Cas9 system further facilitated the targeted knock-in of a protein tag provided by a donor oligonucleotide with knock-in efficiencies of 3.5-15.6%. Mutation rates at potential off-target sites are only 1.1-2.5%, demonstrating the specificity of the CRISPR/Cas9 system. The ease and efficiency of the CRISPR/Cas9 system with limited off-target effects make it a powerful genome engineering tool for in vivo studies.
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Evolution of CRISPR/Cas Systems for Precise Genome Editing.
The bacteria-derived CRISPR/Cas (an acronym for regularly interspaced short palindromic repeats/CRISPR-associated protein) system is currently the most widely used, versatile, and convenient tool for genome engineering. CRISPR/Cas-based technologies have been applied to disease modeling, gene therapies, transcriptional modulation, and diagnostics. Nevertheless, some challenges remain, such as the risk of immunological reactions or off-target effects. To overcome these problems, many new methods and CRISPR/Cas-based tools have been developed. In this review, we describe the current classification of CRISPR systems and new precise genome-editing technologies, summarize the latest applications of this technique in several fields of research, and, finally, discuss CRISPR/Cas system limitations, ethical issues, and challenges.
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Concurrent genome and epigenome editing by CRISPR-mediated sequence replacement.
BACKGROUND: Recent advances in genome editing have facilitated the direct manipulation of not only the genome, but also the epigenome. Genome editing is typically performed by introducing a single CRISPR/Cas9-mediated double-strand break (DSB), followed by non-homologous end joining (NHEJ)- or homology-directed repair-mediated repair. Epigenome editing, and in particular methylation of CpG dinucleotides, can be performed using catalytically inactive Cas9 (dCas9) fused to a methyltransferase domain. However, for investigations of the role of methylation in gene silencing, studies based on dCas9-methyltransferase have limited resolution and are potentially confounded by the effects of binding of the fusion protein. As an alternative strategy for epigenome editing, we tested CRISPR/Cas9 dual cutting of the genome in the presence of in vitro methylated exogenous DNA, with the aim of driving replacement of the DNA sequence intervening the dual cuts via NHEJ. RESULTS: In a proof of concept at the HPRT1 promoter, successful replacement events with heavily methylated alleles of a CpG island resulted in functional silencing of the HPRT1 gene. Although still limited in efficiency, our study demonstrates concurrent epigenome and genome editing in a single event. CONCLUSIONS: This study opens the door to investigations of the functional consequences of methylation patterns at single CpG dinucleotide resolution. Our results furthermore support the conclusion that promoter methylation is sufficient to functionally silence gene expression.
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Guarding the genome: strategies for off-target detection and minimization in CRISPR/Cas9 system.
INTRODUCTION: CRISPR-based genome editing has revolutionized molecular diagnostics and therapeutic applications, offering precise genetic modifications. However, off-target effects remain a significant challenge, potentially leading to unintended mutations and genomic instability. Addressing these concerns is crucial for ensuring the clinical safety and efficacy of CRISPR technologies. AREAS COVERED: This review provides a comprehensive analysis of strategies for detecting and mitigating off-target effects in CRISPR genome editing. We discuss in silico, in vitro, and in vivo detection methods, highlighting their strengths and limitations. Additionally, we examine advancements in guide RNA (gRNA) engineering, improved Cas variants, optimized delivery systems, base and prime editing technologies, and anti-CRISPR proteins as key approaches to enhance genome-editing precision. EXPERT OPINION: While significant progress has been made in off-target detection and mitigation, challenges remain in accurately assessing unintended genomic modifications in vivo and ensuring long-term safety in clinical applications. Future research should focus on developing high-fidelity CRISPR variants, refining computational prediction models, and integrating multi-modal detection systems. Advancing delivery mechanisms and regulatory strategies will be essential for translating CRISPR into safe and effective therapeutic interventions.
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