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A CRISPR/Cas9-based strategy to simultaneously inactivate the entire ALS gene family in Candida orthopsilosis.
Aim: In this study, the CRISPR gene-editing approach was used to simultaneously inactivate all three members of the ALS gene family in the opportunistic pathogen Candida orthopsilosis. Materials & methods: Using a single gRNA and repair template, CRISPR-edited clones were successfully generated in a one-step process in both C. orthopsilosis reference and clinical strains. Results: The phenotypic characterization of the ALS triple-edited strains revealed no impact on growth in liquid or solid media. However, pseudohyphal formation and the ability to adhere to human buccal epithelial cells were significantly decreased in triple-edited clones. Conclusion: Our CRISPR/Cas9 system is a powerful tool for simultaneous editing of fungal gene families, which greatly accelerates the generation of multiple gene-edited Candida strains. Data deposition: Nucleotide sequence data are available in the GenBank databases under the accession numbers MK875971, MK875972, MK875973, MK875974, MK875975, MK875976, MK875977.
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A large-scale genome and transcriptome sequencing analysis reveals the mutation landscapes induced by high-activity adenine base editors in plants.
BACKGROUND: The high-activity adenine base editors (ABEs), engineered with the recently-developed tRNA adenosine deaminases (TadA8e and TadA9), show robust base editing activity but raise concerns about off-target effects. RESULTS: In this study, we perform a comprehensive evaluation of ABE8e- and ABE9-induced DNA and RNA mutations in Oryza sativa. Whole-genome sequencing analysis of plants transformed with four ABEs, including SpCas9n-TadA8e, SpCas9n-TadA9, SpCas9n-NG-TadA8e, and SpCas9n-NG-TadA9, reveal that ABEs harboring TadA9 lead to a higher number of off-target A-to-G (A>G) single-nucleotide variants (SNVs), and that those harboring CRISPR/SpCas9n-NG lead to a higher total number of off-target SNVs in the rice genome. An analysis of the T-DNAs carrying the ABEs indicates that the on-target mutations could be introduced before and/or after T-DNA integration into plant genomes, with more off-target A>G SNVs forming after the ABEs had integrated into the genome. Furthermore, we detect off-target A>G RNA mutations in plants with high expression of ABEs but not in plants with low expression of ABEs. The off-target A>G RNA mutations tend to cluster, while off-target A>G DNA mutations rarely clustered. CONCLUSION: Our findings that Cas proteins, TadA variants, temporal expression of ABEs, and expression levels of ABEs contribute to ABE specificity in rice provide insight into the specificity of ABEs and suggest alternative ways to increase ABE specificity besides engineering TadA variants.
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Rapid and efficient CRISPR/Cas9 gene editing in Citrus using the YAO promoter.
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Genetic editing of the virulence gene of Escherichia coli using the CRISPR system.
Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 is an emerging gene-editing technology that is widely used in prokaryotes and eukaryotes. It can realize the specific manipulation of the genome efficiently and accurately. CRISPR/Cas9 coupled λ-Red recombination technology was used to perform genome editing in different genes. For finding an efficient method to edit the virulence genes of enterotoxigenic E. coli (ETEC), the two-plasmid system was used. The coding sequence (CDS) region of the estA, eltI, estB, eltIIc1, and faeG locus were deleted. The coding region of estB was substituted with estA. Gene recombination efficiency ranged from 0 to 77.78% when the length of the homology arm was from 50 to 300 bp. Within this range, the longer the homology arm, the higher the efficiency of genetic recombination. The results showed that this system can target virulence genes located in plasmids and on chromosomes of ETEC strains. A single base mutation was performed by two-step gene fragment replacement. This study lays the foundation for research on virulence factors and genetic engineering of vaccines for ETEC.
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Extended pegRNAs enhance the editing capability of Prime editing.
Genome editing is highly valuable in biomedical research. Despite their versatility, current Prime editing (PE) techniques are limited to short sequence alterations [up to ~44 base pairs (bp)], and exhibit inconsistent or low efficiency across genomic loci, particularly when faced with poly-T sequences. To address these challenges, we developed an extended PE (exPE) technique that can potentially execute any precise genome editing. By harnessing RNA polymerase II (Pol II) promoters to transcribe extended PE guide RNAs (expegRNAs), exPE substantially improves editing efficiency and overcomes the challenges posed by poly-T sequences. Compared with conventional PE, exPE achieves up to a 14-fold increase in the efficiency of base conversions and short insertions, and, remarkably, up to a 259-fold improvement in regions with poly-T sequences. Uniquely, exPE enables seamless insertion of gene-sized DNA fragments into genomes, potentially correcting nearly 90% of human genetic variants, thereby broadening its applications in genetic research and therapy.
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CircRNA knockout/knockdown tools in molecular biology research.
Circular RNAs (circRNAs), characterized by their covalently closed circular architecture, represent a unique class of endogenous RNA molecules that serve as pivotal regulators in post-transcriptional gene regulation in organisms. Accumulating evidence has established their potential as promising diagnostic biomarkers across various human pathologies, including but not limited to malignant neoplasms, neurodegenerative disorders, and metabolic dysregulation.By inhibiting circRNA expression, we can better understand their functions and their impact on related biological processes. Over the past decade, remarkable advancements have emerged in circRNAs manipulation technologies, including siRNA, DNAzyme, and CRISPR-Cas systems emerging as powerful tools for precise circRNAs editing in both experimental models and preclinical studies. In this review, we summarize the advantages and identification of these editing methods and discuss future challenges and prospects.
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Next-generation gene editing strategies in cancer: Integrating CRISPR, PROTACs, and advanced molecular technologies.
Cancer is a significant therapeutic problem as tumors are heterogeneous, multidrug-resistant, and oncogenic drivers are undruggable. Genome editing and targeted protein degradation are emerging approaches that are transforming precision oncology by allowing genetic and proteomic interventions. Such technologies as CRISPR-based systems and proteolysis-targeting chimeras (PROTACs) are alternative methods of correcting genes and the selective removal of a protein. Their combination provides them with new possibilities regarding long-lasting and specific cancer treatment and discovery of new therapeutic targets. Although this has good news, there are delivery, off-target effects and safety challenges. The continued advancements in nanotechnology, artificial intelligence (AI), and personalized medicine will be likely to improve clinical translation. Generally, the integration of these technologies is an inducing trend in the treatment of cancer in the next generation.
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CRISPR-Cas technologies: Emerging tools from research to clinical application.
CRISPR-Cas technologies have emerged as powerful and versatile tools in gene therapy. In addition to the widely used SpCas9 system, alternative platforms including modified amino acid sequences, size-optimized variants, and other Cas enzymes from diverse bacterial species have been developed to apply this technology in various genetic contexts. In addition, base editors and prime editors for precise gene editing, the Cas13 system targeting RNA, and CRISPRa/i systems have enabled diverse and adaptable approaches for genome and RNA editing, as well as for regulating gene expression. Typically, CRISPR-Cas components are transported to the target in the form of DNA, RNA, or ribonucleoprotein complexes using various delivery methods, such as electroporation, adeno-associated viruses, and lipid nanoparticles. To amplify therapeutic efficiency, continued developments in targeted delivery technologies are required, with increased safety and stability of therapeutic biomolecules. CRISPR-based therapeutics hold an inexhaustible potential for the treatment of many diseases, including rare congenital diseases, by making permanent corrections at the genomic DNA level. In this review, we present various CRISPR-based tools, their delivery systems, and clinical progress in the CRISPR-Cas technology, highlighting its innovative prospects for gene therapy.
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Effective generation of maternal genome point mutated porcine embryos by injection of cytosine base editor into germinal vesicle oocytes.
Cytosine and adenine base editors are promising new tools for introducing precise genetic modifications that are required to generate disease models and to improve traits in pigs. Base editors can catalyze the conversion of C→T (C>T) or A→G (A>G) in the target site through a single guide RNA. Injection of base editors into the zygote cytoplasm can result in the production of offspring with precise point mutations, but most F0 are mosaic, and breeding of F1 heterozygous pigs is time-intensive. Here, we developed a method called germinal vesicle oocyte base editing (GVBE) to produce point mutant F0 porcine embryos by editing the maternal alleles during the GV to MII transition. Injection of cytosine base editor 3 (BE3) mRNA and X-linked Dmd-specific guide RNAs into GVoocytes efficiently edited maternal Dmd during in vitro maturation and did not affect the maturation potential of the oocytes. The edited MII oocytes developed into blastocysts after parthenogenetic activation (PA) or in vitro fertilization (IVF). However, BE3 may reduce the developmental potential of IVF blastocysts from 31.5%±0.8% to 20.4% ±2.1%. There 40%-78.3% diploid PA blastocysts had no more than two different alleles, including up to 10% embryos that had only C>T mutation alleles. Genotyping of IVF blastocysts indicated that over 70% of the edited embryos had one allele or two different alleles of Dmd. Since the male embryos had only a copy of Dmd allele, all five (5/19) F0 male embryos are homozygous and three of them were Dmd precise C>T mutation. Nine (9/19) female IVF embryos had two different alleles including a WT and a C>T mutation. DNA sequencing showed that some of them might be heterozygous embryos. In conclusion, the GVBE method is a valuable method for generating F0 embryos with maternal point mutated alleles in a single step.
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Construction of a CRISPR-Cas9-Based Genetic Editing Tool for Serratia marcescens Using a Stationary Phase Promoter and Its Application in Putrescine Production.
Putrescine plays a significant role in green food production and agriculture by promoting plant growth and enhancing crop quality. Its application reduces the reliance on chemical fertilizers and pesticides, thereby supporting the advancement of sustainable agricultural practices. This study achieved efficient production of putrescine in Serratia marcescens. S. marcescens has been extensively used to synthesize antimicrobial substances and express proteins, but its application has been limited by the lack of efficient genome-editing tools. This study presents a CRISPR-Cas9-based tool for gene editing in S. marcescens. A dual-plasmid system was constructed, incorporating an editing template into the plasmid pEdit with target-specific sgRNA. A stationary phase promoter was used to express Cas9 from Streptococcus pyogenes protein, avoiding the need for additional inducers and ensuring efficient one-step gene knockout and integration. The tool demonstrated over 80% editing efficiency across various S. marcescens strains and enabled successful single-base mutations. Using this tool, we enhanced putrescine production in S. marcescens HBQA7, optimizing the expression of ornithine decarboxylase from Clostridium aceticum DSM1496 with the P2 promoter and identifying the optimal integration site. Putrescine production reached 8.46 g/L within 48 h. This study significantly advances S. marcescens gene editing and metabolic engineering.
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