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[Experimental Selection of Effective sgRNAs for MmCas12m Targeting the Region of the Start Codon of the HIV-1 gag Gene].
HIV-1 remains a threat to global health. There is no effective vaccine or drug for a complete cure of HIV infection. Work continues on the development of gene therapy drugs against HIV-1. The use of DNA base editors delivered to the editing site by CRISPR-Cas systems has shown some success in the field of HIV-1 gene therapy. The MmCas12m isoform obtained from Mycolicibacterium mucogenicum can become a promising platform for this task. MmCas12m has a compact size, the ability to bind strongly to the target DNA sequence, and an absence of nuclease activity. Thus, MmCas12m can act not only as a platform for the delivery of DNA base editors, but also as an inhibitor of transcription from HIV-1 proviral DNA. We experimentally selected in vitro the most effective sgRNAs for MmCas12m to target the start codon of the gag HIV-1, the product of which is important in virion assembly. Of the nine sgRNAs we selected, four showed statistically significant effectiveness in targeting the desired region of the HIV-1 genome. To test the effectiveness of each sgRNA, we have developed a system suitable for evaluating the binding of any Cas protein to the target site of HIV-1 genome editing.
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T cell-specific inactivation of mouse CD2 by CRISPR/Cas9.
The CRISPR/Cas9 system can be used to mutate target sequences by introduction of double-strand breaks followed by imprecise repair. To test its use for conditional gene editing we generated mice transgenic for CD4 promoter-driven Cas9 combined with guide RNA targeting CD2. We found that within CD4(+) and CD8(+) lymphocytes from lymph nodes and spleen 1% and 0.6% were not expressing CD2, respectively. T cells lacking CD2 carryied mutations, which confirmed that Cas9 driven by cell-type specific promoters can edit genes in the mouse and may thus allow targeted studies of gene function in vivo.
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Double-stranded DNA deaminase DddAE1347A can increase the efficiency and targeting range of cytidine base editors.
BACKGROUND: Cytidine base editors (CBEs) consist of a single-strand specific cytidine deaminase fused to Cas9 nickase, enabling efficient C-to-T conversion across diverse organisms. Enhancing editing range and efficiency of these tools is essential for expanding their applications. RESULTS: In this study, we report that fusing a double-stranded DNA-specific cytosine deaminase DddAE1347A to CBEs significantly improves editing activity and broadens the editing window in cell lines, embryos, tobacco, and cotton. Compared to BE4max, the optimized DddAE1347A-BE4max exhibits up to a 93- fold increase in editing efficiency, achieving up to 52% efficiency at C14 and C15 in cell lines. Further investigation reveals that DddAE1347A is compatible with various Cas9 variants (SpCas9, SpaCas9, and Nme2Cas9) and deaminase variants (rA1, A3G, and A3A). Additionally, we demonstrate that cytosine deaminases with single-stranded DNA activity fail to enhance the CBE system. In contrast, various DddA variants can improve CBE editing activity at PAM-proximal cytosine positions, highlighting the modularity of fusion between DddAs and CBEs. CONCLUSIONS: These findings suggest that the double-stranded DNA-specific cytosine deaminase protein can act as an engineered fusion module in the CBE system, altering the performance (window/efficiency) of CBEs.
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Integrating genomics and genome editing for orphan crop improvement: a bridge between orphan crops and modern agriculture system.
Domestication of orphan crops could be explored by editing their genomes. Genome editing has a lot of promise for enhancing agricultural output, and there is a lot of interest in furthering breeding in orphan crops, which are sometimes plagued with unwanted traits that resemble wild cousins. Consequently, applying model crop knowledge to orphan crops allows for the rapid generation of targeted allelic diversity and innovative breeding germplasm. We explain how plant breeders could employ genome editing as a novel platform to accelerate the domestication of semi-domesticated or wild plants, resulting in a more diversified base for future food and fodder supplies. This review emphasizes both the practicality of the strategy and the need to invest in research that advances our understanding of plant genomes, genes, and cellular systems. Planting more of these abandoned orphan crops could help alleviate food scarcities in the challenge of future climate crises.
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Streamlined CRISPR genome engineering in wild-type bacteria using SIBR-Cas.
CRISPR-Cas is a powerful tool for genome editing in bacteria. However, its efficacy is dependent on host factors (such as DNA repair pathways) and/or exogenous expression of recombinases. In this study, we mitigated these constraints by developing a simple and widely applicable genome engineering tool for bacteria which we termed SIBR-Cas (Self-splicing Intron-Based Riboswitch-Cas). SIBR-Cas was generated from a mutant library of the theophylline-dependent self-splicing T4 td intron that allows for tight and inducible control over CRISPR-Cas counter-selection. This control delays CRISPR-Cas counter-selection, granting more time for the editing event (e.g. by homologous recombination) to occur. Without the use of exogenous recombinases, SIBR-Cas was successfully applied to knock-out several genes in three wild-type bacteria species (Escherichia coli MG1655, Pseudomonas putida KT2440 and Flavobacterium IR1) with poor homologous recombination systems. Compared to other genome engineering tools, SIBR-Cas is simple, tightly regulated and widely applicable for most (non-model) bacteria. Furthermore, we propose that SIBR can have a wider application as a simple gene expression and gene regulation control mechanism for any gene or RNA of interest in bacteria.
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CRISPR/Cas9 mediated targeting of multiple genes in Dictyostelium.
CRISPR/Cas9 has emerged in various organisms as a powerful technology for targeted gene knockout; however, no reports of editing the Dictyostelium genome efficiently using this system are available. We describe here the application of CRISPR/Cas9-mediated gene modification in Dictyostelium. The endogenous tRNA-processing system for expressing sgRNA was approximately 10 times more effective than the commonly used U6 promoter. The resulting sgRNA affected the sub-nuclear localisation of Cas9, indicating that the expression level of sgRNA was sufficiently high to form Cas9 and sgRNA complexes within the nucleus. The all-in-one vector containing Cas9 and sgRNA was transiently expressed to generate mutants in five PI3K genes. Mutation detective PCR revealed the mutagenesis frequency of the individual genes to be between 72.9% and 100%. We confirmed that all five targeting loci in the four independent clones had insertion/deletion mutations in their target sites. Thus, we show that the CRISPR/Cas9 system can be used in Dictyostelium cells to enable efficient genome editing of multiple genes. Since this system utilises transient expression of the all-in-one vector, it has the advantage that the drug resistance cassette is not integrated into the genome and simple vector construction, involving annealing two oligo-DNAs.
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Effects of CRISPR technology on agricultural sustainability: global applications and turkish perspective.
This review evaluates CRISPR/Cas applications in agriculture from a global perspective with explicit reference to Türkiye. Using a literature gap-matrix approach organised around four analytical dimensions-environmental, economic, social and policy, and scientific and technological-we synthesize the primary evidence on water and input use, productivity, disease resistance, and product quality. The literature concentrates on water and fertilizer use, productivity, and off-target accuracy, whereas soil health, biodiversity, consumer acceptance, ethical considerations and regulatory frameworks remain systematically under-represented. Global deployment of CRISPR is already delivering measurable advantages in food security, shelf life and nutritional value, while in Türkiye the research base is at an early stage but has clear potential in wheat, barley, tomato and olive. Translating CRISPR into Turkish agricultural sustainability requires (i) a domestic biosafety framework aligned with the emerging European New Genomic Techniques approach, (ii) sustained investment in multi-location primary field trials, and (iii) inclusive deployment mechanisms-particularly through producer cooperatives-that allow smallholder farmers to benefit from edited varieties.
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Perfectly matched 20-nucleotide guide RNA sequences enable robust genome editing using high-fidelity SpCas9 nucleases.
High-fidelity SpCas9 variants (eSpCas9 and SpCas9-HF1) have been engineered to reduce off-target effects. We found that changes in guide RNA length induced significant reductions in the editing activities of SpCas9 variants in plant cells. Single guide RNAs harboring precise, perfectly matched 20-nucleotide guide sequences are necessary for high on-target editing activities of eSpCas9 and SpCas9-HF1. Precise 20-nucleotide guide sequences derived from tRNA-sgRNA precursors enable robust on-target editing by these variants with enhanced specificity. Our work reveals an effective way of enhancing the use of the high-fidelity SpCas9 nucleases for efficient and precise genome engineering.
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Unexpected heterogeneity derived from Cas9 ribonucleoprotein-introduced clonal cells at the HPRT1 locus.
Single-cell cloning is an essential technique for establishing genome-edited cell clones mediated by programmable nucleases such as CRISPR-Cas9. However, residual genome-editing activity after single-cell cloning may cause heterogeneity in the clonal cells. Previous studies showed efficient mutagenesis and rapid degradation of CRISPR-Cas9 components in cultured cells by introducing Cas9 ribonucleoproteins (RNPs). In this study, we investigated how the timing for single-cell cloning of Cas9 RNP-transfected cells affected the heterogeneity of the resultant clones. We carried out transfection of Cas9 RNPs targeting several loci in the HPRT1 gene in HCT116 cells, followed by single-cell cloning at 24, 48, 72 hr and 1 week post-transfection. After approximately 3 weeks of incubation, the clonal cells were collected and genotyped by high-resolution microchip electrophoresis and Sanger sequencing. Unexpectedly, long-term incubation before single-cell cloning resulted in highly heterogeneous clones. We used a lipofection method for transfection, and the media containing transfectable RNPs were not removed before single-cell cloning. Therefore, the active Cas9 RNPs were considered to be continuously incorporated into cells during the precloning incubation. Our findings provide a warning that lipofection of Cas9 RNPs may cause continuous introduction of gene mutations depending on the experimental procedures.
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Regulation of Single and Multiple Genes in Bacillus amyloliquefaciens by an Evolution System In Vivo.
With the development of synthetic biology, an evolution system in vivo has been applied to accelerate the construction of cell factories. In this study, an efficient in vivo evolution system was developed for regulation of single and multiple genes in Bacillus amyloliquefaciens. First, the CRISPR/Cas9n-AID base editor was constructed through integration expression of the fused Cas9n protein and activation-induced cytidine deaminase (AID), and the base conversion efficiency from C to T was as high as 90% in single-gene editing. Subsequently, the evolution template (XP43) with an editable RBS sequence (GGGGGGGG) was designed for in vivo evolution through two strategies. By next-generation sequencing of RBS mutation libraries, the extended sgRNA strategy was confirmed to be the optimal evolution scheme. Using the alkaline protease gene (aprE) as the single gene target, the evolution program was initiated to successfully obtain a series of mutant strains with gradient AprE activities. Furthermore, multiple key genes (dhemA, SAM2, and hemEHY) were evolved simultaneously to balance the heme metabolic network, and the optimal mutant strain (HZHA-C2) produced 14.02 mg/L heme, 93% higher than the control strain. Finally, the overexpression of the hemH gene further increased the heme titer by 49%. By a fed-batch fermentation strategy, the heme titer of the optimal engineered strain (HZHA2/pHY-hemH) was improved by 64%, achieving 32.61 mg/L.
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