A potent EF1A promoter and a minimal U6 promoter enable highly efficient CRISPR genome editing in Aedes aegypti.

A stable and potent buffalo EF1α1 promoter for robust gene expression in mammalian systems

Improving the efficiency of CRISPR/Cas9-induced multiplex editing in Brassica napus L.

CRISPR/Cas9 is currently the most powerful genome editing tool for crop improvement and gene function exploration. However, the mutation efficiency of CRISPR/Cas9 in Brassica napus, a globally important allotetraploid oil crop, so far achieved remains significant potential for further improvement compared to that observed in model species. In this study, we systematically evaluated three key factors influencing editing efficiency: sgRNA promoters, sgRNA structural modifications, and Cas9-driven promoters. First, we compared all sgRNA promoters currently used in B. napus side by side. The average editing efficiency of AtU6–26 was the highest (44.74%), followed by AtU3b (36.18%), AtU6–29 (28.49%), AtU3d (17.73%) and AtU6–1 (0.53%). Second, we demonstrated that enhanced sgRNA (esgRNA) enhanced CRISPR/Cas9 editing efficiency by 52.1% compared to native sgRNA. The combination of AtU6–26 with esgRNA achieved the highest editing efficiency (average 61.4%). Third, we identified three endogenous promoters (pCAB1, pLTP2, and pTCTP) with high expression levels in the hypocotyls and callus, to replace the 35S promoter of Cas9. The CAB1 promoter was the most efficient and significantly enhanced the mutation efficiency by 31.46% over the commonly used 35S promoter. Thus, these optimizations provide valuable strategies for improving CRISPR/Cas9 efficiency in Brassica crops.

Targeted Gene Expression Modulation Using CRISPR/dCas9 to Investigate Pathogenic Outcomes in Tomato.

A Novel, Simple, and Highly Efficient Dual-sgRNA CRISPR/Cas9-Based Editing System in the Filamentous Fungus Fusarium oxysporum

Fusarium oxysporum, as one of the most common filamentous fungi, possesses great biosynthetic potential for natural products. However, the lack of efficient genetic tools has hindered functional genome mining and metabolic engineering in this fungus. In this study, a novel highly efficient CRISPR/Cas9-based dual-sgRNA expression editing system for F. oxysporum was successfully developed through construction of a robust plasmid platform pFRCas9-G418 using incorporation of an endogenous histone H2B nuclear localization signal and a 5S rRNA promoter-driven polycistronic tRNA−sgRNA cassette. This system is suitable not only for single-gene editing but also for large-fragment deletion and multiplex gene editing, although the editing efficiency is somewhat lower. First, this new CRISPR/Cas9 system exhibited a high efficiency of 93.75% ± 6.25% for deletion of the Fusarium cyclin C1 (fcc1) gene (∼1 kb), which was usually selected as the target gene responsible for yellow pigment accumulation. Then, knockout of the core NRPS gene sanB (∼19 kb) and knock-in of the strong promoter gpdA in the N-methylsansalvamide (SA) biosynthetic gene cluster (BGC) in strain F. oxysporum R1 using this system, respectively, led to no SA yield and an increase of 26.4% SA titer, confirming its capacity for large gene deletion and gene knock-in. Furthermore, one-step dual-gene knockout of hat1 (histone acetyltransferase gene, ∼1.5 kb) and pacC (pH-responsive transcription factor, ∼2 kb) was first achieved in Fusarium species. This versatile platform provides a powerful tool for editing gene(s) of various sizes in F. oxysporum.

Finding the perfect promoter for Cas9 in homing gene drives using single cell transcriptome data

Gene drive can modify or suppress vector populations by spreading drive alleles. In CRISPR homing drives, regulating Cas9 expression has been effective for improving drive performance, but selecting suitable promoters is often a major challenge. Here, we evaluate 35 Cas9 constructs with distinct promoters in Drosophila melanogaster and identify associations between drive performance and single-cell RNA expression patterns of the promoter-associated genes. Our results indicate that higher drive conversion is associated with elevated expression of the promoter-associated gene in reproductive cells, but embryo resistance allele formation correlates with excessive female germline expression. For males, early germline expression produces superior performance. Thus, optimal drive performance requires restricting Cas9 expression to a tight quantitative and spatiotemporal window. Additionally, we find that an in situ construct significantly reduces potentially harmful somatic expression. Based on these results, we propose criteria for selecting promoters, providing a rationale and guidance for optimization of homing gene drives. CRISPR homing gene drives can help control disease vectors but require precise Cas9 expression. Here, the drive performance of 35 Cas9 constructs with different promoters in Drosophila were compared to single cell RNA expression patterns of promoter-associated genes.

Establishment of an efficient Agrobacterium-mediated transformation system and CRISPR/Cas9-mediated genome editing of the OvPDS1 gene in Oxalis vulcanicola ‘Sunset Velvet’