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A Cloning-Free Method for CRISPR/Cas9-Mediated Genome Editing in Fission Yeast.
The CRISPR/Cas9 system, which relies on RNA-guided DNA cleavage to induce site-specific DNA double-strand breaks, is a powerful tool for genome editing. This system has been successfully adapted for the fission yeast Schizosaccharomyces pombe by expressing Cas9 and the single-guide RNA (sgRNA) from a plasmid. In the procedures published to date, the cloning step that introduces a specific sgRNA target sequence into the plasmid is the most tedious and time-consuming. To increase the efficiency of applying the CRISPR/Cas9 system in fission yeast, we here developed a cloning-free procedure that uses gap repair in fission yeast cells to assemble two linear DNA fragments, a gapped Cas9-encoding plasmid and a PCR-amplified sgRNA insert, into a circular plasmid. Both fragments contain only a portion of the ura4 or bsdMX marker so that only the correctly assembled plasmid can confer uracil prototrophy or blasticidin resistance. We show that this gap-repair-based and cloning-free CRISPR/Cas9 procedure permits rapid and efficient point mutation knock-in, endogenous N-terminal tagging, and genomic sequence deletion in fission yeast.
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Guide RNA Design for CRISPR/Cas9-Mediated Potato Genome Editing.
The activity of the pool of sgRNA molecules designed for different regions of potato coilin and phytoene desaturase genes was compared in vitro. Due to the presence of nucleotides unpaired with DNA, sgRNA is able not only to inhibit but also to stimulate the activity of the Cas9-sgRNA complex in vitro. Although the first six nucleotides located in the DNA substrate proximally to the PAM site at the 3' end are the binding sites for cas9, they had no significant effect on the activity of the Cas9-sgRNA complex.
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Coloring target and off-target effects of genetically modified nucleases by blue & white colony assays.
More sensitive evaluation of the off-target effects of gene editing nucleases is crucial for human gene therapy. Here we report chromogenic assays designed for sensitive evaluation of gene editing activities using CRISPR/Cas9 test system. Based on beta-galactosidase alpha complementation, qualitative and quantitative evaluations of the target and off-target effects of CRISPR/Cas9 were well established through the color alteration of the E.coli colonies. In addition to target effect analysis, these new assays provide extremely sensitive and efficient tool to profile the off-target effects with one or more bases mismatched between the targets and the gRNAs. Moreover, these assays allow the identification of gene editing effects for off-targets with one base mismatched PAM sites.
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Enhanced Genome Editing Activity with Novel Chimeric ScCas9 Variants in Rice.
The Streptococcus canis Cas9 protein (ScCas9) recognizes the NNG protospacer adjacent motif (PAM), offering a wider range of targets than that offered by the commonly used S. pyogenes Cas9 protein (SpCas9). However, both ScCas9 and its evolved Sc++ variant still exhibit low genome editing efficiency in plants, particularly at the less preferred NTG and NCG PAM targets. In this study, a chimeric SpcRN++ variant is engineered by grafting the recognition (REC) domain of SpCas9 into the Sc++ variant, incorporating the R221K/N394K mutations, and retaining the positively charged loop of S. anginosus Cas9. The SpcRN++ variant exhibits a higher genome editing capacity and wider target range than the Sc++ variant in rice protoplasts and stable transgenic plants. Further evidence indicates that nSpcRN++-based A3A/Y130F and TadA8e exhibit enhanced cytosine and adenine editing efficiency in plants. Finally, herbicide-resistant rice germplasms are produced by targeting the OsACC gene using nSpcRN++-based adenine base editors. These results demonstrate that SpcRN++ is a powerful tool for genome editing in plants, and this integrative protein engineering strategy holds promise for engineering other Cas9 proteins.
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Structure-optimized sgRNA selection with PlatinumCRISPr for efficient Cas9 generation of knockouts.
A single guide RNA (sgRNA) directs Cas9 nuclease for gene-specific scission of double-stranded DNA. High Cas9 activity is essential for efficient gene editing to generate gene deletions and gene replacements by homologous recombination. However, cleavage efficiency is below 50% for more than half of randomly selected sgRNA sequences in human cell culture screens or model organisms. We used in vitro assays to determine intrinsic molecular parameters for maximal sgRNA activity including correct folding of sgRNAs and Cas9 structural information. From the comparison of over 10 data sets, we find major constraints in sgRNA design originating from defective secondary structure of the sgRNA, sequence context of the seed region, GC context, and detrimental motifs, but we also find considerable variation among different prediction tools when applied to different data sets. To aid selection of efficient sgRNAs, we developed web-based PlatinumCRISPr, an sgRNA design tool to evaluate base-pairing and sequence composition parameters for optimal design of highly efficient sgRNAs for Cas9 genome editing. We applied this tool to select sgRNAs to efficiently generate gene deletions in Drosophila Ythdc1 and Ythdf, that bind to N 6 methylated adenosines (m6A) in mRNA. However, we discovered that generating small deletions with sgRNAs and Cas9 leads to ectopic reinsertion of the deleted DNA fragment elsewhere in the genome. These insertions can be removed by standard genetic recombination and chromosome exchange. These new insights into sgRNA design and the mechanisms of CRISPR-Cas9 genome editing advance the efficient use of this technique for safer applications in humans.
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Detection of on-target and off-target mutations generated by CRISPR/Cas9 and other sequence-specific nucleases.
The development of customizable sequence-specific nucleases such as TALENs, ZFNs and the powerful CRISPR/Cas9 system has revolutionized the field of genome editing. The CRISPR/Cas9 system is particularly versatile and has been applied in numerous species representing all branches of life. Regardless of the target organism, all researchers using sequence-specific nucleases face similar challenges: confirmation of the desired on-target mutation and the detection of off-target events. Here, we evaluate the most widely-used methods for the detection of on-target and off-target mutations in terms of workflow, sensitivity, strengths and weaknesses.
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Development of TALE-adenine base editors in plants.
Base editors enable precise nucleotide changes at targeted genomic loci without requiring double-stranded DNA breaks or repair templates. TALE-adenine base editors (TALE-ABEs) are genome editing tools, composed of a DNA-binding domain from transcription activator-like effectors (TALEs), an engineered adenosine deaminase (TadA8e), and a cytosine deaminase domain (DddA), that allow A•T-to-G•C editing in human mitochondrial DNA. However, the editing ability of TALE-ABEs in plants apart from chloroplast DNA has not been described, so far, and the functional role how DddA enhances TadA8e is still unclear. We tested a series of TALE-ABEs with different deaminase fusion architectures in Nicotiana benthamiana and rice. The results indicate that the double-stranded DNA-specific cytosine deaminase DddA can boost the activities of single-stranded DNA-specific deaminases (TadA8e or APOBEC3A) on double-stranded DNA. We analysed A•T-to-G•C editing efficiencies in a β-glucuronidase reporter system and showed precise adenine editing in genomic regions with high product purity in rice protoplasts. Furthermore, we have successfully regenerated rice plants with A•T-to-G•C mutations in the chloroplast genome using TALE-ABE. Consequently, TALE-adenine base editors provide alternatives for crop improvement and gene therapy by editing nuclear or organellar genomes.
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Affordable CRISPR RNP-Based Genome Editing in Euglena gracilis.
Genome editing can enhance basic research and enable industrial applications of green algae. Here, we present an affordable, broadly applicable workflow for genome editing in the unicellular green alga Euglena gracilis using Cas9 nucleases. This method retains high editing efficiency while significantly lowering technical barriers. Unlike previous approaches that required specialized equipment, this protocol can be performed using a general-purpose laboratory electroporator and a simplified clonal isolation procedure without the need for specialized micromanipulation devices. This protocol is compatible with a range of editing outcomes, such as targeted deletions and precise base substitutions, enabling more widespread genome editing in Euglena. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Culture of Euglena gracilis Basic Protocol 2: sgRNA synthesis Basic protocol 3: Transformation Basic protocol 4: Genotyping.
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CRISPR-Cas9 vectors for genome editing and host engineering in the baculovirus-insect cell system.
The baculovirus-insect cell system (BICS) has been widely used to produce many different recombinant proteins for basic research and is being used to produce several biologics approved for use in human or veterinary medicine. Early BICS were technically complex and constrained by the relatively primordial nature of insect cell protein glycosylation pathways. Since then, recombination has been used to modify baculovirus vectors-which has simplified the system-and transform insect cells, which has enhanced its protein glycosylation capabilities. Now, CRISPR-Cas9 tools for site-specific genome editing are needed to facilitate further improvements in the BICS. Thus, in this study, we used various insect U6 promoters to construct CRISPR-Cas9 vectors and assessed their utility for site-specific genome editing in two insect cell lines commonly used as hosts in the BICS. We demonstrate the use of CRISPR-Cas9 to edit an endogenous insect cell gene and alter protein glycosylation in the BICS.
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Engineering Nucleotidoproteins for Base-Pairing-Assisted Cytosolic Delivery and Genome Editing.
Protein therapeutics targeting intracellular machineries hold profound potential for disease treatment, and hence robust cytosolic protein delivery technologies are imperatively demanded. Inspired by the super-negatively charged, nucleotide-enriched structure of nucleic acids, adenylated pro-proteins (A-proteins) with dramatically enhanced negative surface charges have been engineered for the first time via facile green synthesis. Then, thymidine-modified polyethyleneimine is developed, which exhibits strong electrostatic attraction, complementary base pairing, and hydrophobic interaction with A-proteins to form salt-resistant nanocomplexes with robust cytosolic delivery efficiencies. The acidic endolysosomal environment enables traceless restoration of the A-proteins and consequently promotes the intracellular release of the native proteins. This strategy shows high efficiency and universality for a variety of proteins with different molecular weights and isoelectric points in mammalian cells. Moreover, it enables highly efficient delivery of CRISPR-Cas9 ribonucleoproteins targeting fusion oncogene EWSR1-FLI1, leading to pronounced anti-tumor efficacy against Ewing sarcoma. This study provides a potent and versatile platform for cytosolic protein delivery and gene editing, and may benefit the development of protein pharmaceuticals.
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