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Crosstalk between myostatin and callipyge in CRISPR/Cas9-edited goat fibroblast cells.
Myostatin (MSTN) and Callipyge (CLPG) genes are key regulators of muscle growth. While MSTN inhibits muscle development, the CLPG mutation induces muscle hypertrophy through a specific imprinted genetic mechanism. The interaction between these genes remains of interest for improving livestock muscle traits. In this study, CRISPR/Cas9 was employed to edit MSTN and CLPG genes in goat fibroblast cells via electrotransfection. Cells were selected using puromycin antibiotic, and gene-editing efficiency was evaluated through Sanger sequencing. Gene expression changes were analyzed using RT-qPCR analysis. MSTN gene knockout resulted in significant downregulation of MSTN and CLPG, while GTL2 expression was upregulated by more than 50-fold. Additionally, myosin heavy chain genes (MYH1, MYH3, MYH4) were strongly upregulated, with MYH3 13-fold and MYH4 30-fold increase in the expression. In CLPG-edited cells, the expression of MSTN, TRIM28, and CLPG was reduced, while GTL2 was upregulated by 6-fold. MYH3 and MYH4 expression increased 4-fold in CLPG-edited cells, though the increase was less pronounced compared to MSTN-edited cells. DLK1 expression was undetectable in both non-edited control and gene-edited fibroblast cells. Our findings support the interaction between MSTN and CLPG, contributing to the regulation of muscle growth. Notably, the study also highlights the challenges associated with editing imprinted genes like CLPG and suggests that TRIM28 may play a role downstream of CLPG regulation. These results provide valuable insights into muscle development regulation, offering potential applications in livestock genetic improvement.
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Non-viral delivery of the CRISPR/Cas system: DNA versus RNA versus RNP.
Since its discovery, the CRISPR/Cas technology has rapidly become an essential tool in modern biomedical research. The opportunities to specifically modify and correct genomic DNA have also raised big hope for therapeutic applications by direct in vivo genome editing. In order to achieve the intended genome modifications, the functional unit of the CRISPR/Cas system finally has to be present in the nucleus of target cells. This can be achieved by delivery of different biomolecular Cas9 and gRNA formats: plasmid DNA (pDNA), RNA or Cas9 ribonucleoproteins (RNPs). While the initial research focussed on pDNA transfections, the currently most promising strategy for systemic non-viral in vivo delivery is based on RNA which has achieved remarkable results in the first clinical trials. RNP delivery receives much attention for ex vivo applications, but the translation to systemic in vivo genome editing in patients has not been reached so far. The article summarises the characteristics and differences of each format, provides an overview of the published delivery strategies and highlights recent examples of delivery systems including the status of clinical applications.
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Assessment of the effect of ten heading time genes on reproductive transition and yield components in rice using a CRISPR/Cas9 system.
We demonstrated the effect of heading time genes on reproductive transition and yield components under an identical genetic background using CRISPR/Cas9 gene-editing technology, and we propose that the elite allele will provide a new breeding strategy for rice breeding in high-latitude regions. Heading date is a factor closely associated with grain yield in rice (Oryza sativa L). In recent decades, a number of genes responsible for heading time have been identified, the variation of which contributes to the expansion of the rice cultivation area. However, it is difficult to compare the phenotypic effects of these genes due to the different genetic backgrounds. In this study, we generated 14 heading time mutants using CRISPR/Cas9 gene-editing technology and marker-assisted selection with a japonica Sasanishiki wild-type (WT) genetic background. Photoperiod sensitivity, the relationship between days to heading (DTH), and yield components of mutants were investigated. We found that the yield increases with increases in DTH, but eventually plateaus at maximum and then began to decrease, whereas the biomass continued to increase. The mutants exerted distinctly different effects on DTH and yield components. The convergent double mutants had severe yield reduction compared with single mutants, even with a DTH that was similar to that of single mutants. We also found that an elite mutant of se14 achieved a yield equal to that of the WT, but with heading occurring 10 days earlier. A sequence analysis of 72 cultivars collected from the japonica cultivated zone shows that elite se14 mutants have not been applied to rice breeding. Our study demonstrates the effect of heading time genes on reproductive transition and yield components under an identical genetic background. These results may provide new insights into rice breeding using heading time mutants.
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Comprehensive molecular characterization and biosafety assessment of TaDOF7.6 transgenic and gene-edited wheat.
BACKGROUND: Wheat is one of the major staple food crops worldwide, and stable wheat yield is crucial for ensuring global food security. Transgenic and gene-editing technologies serve as important approaches for wheat genetic improvement, yet further application of related materials requires systematic evaluation of their molecular characteristics, genetic stability and environmental biosafety. In this study, TaDOF7.6-RNAi transgenic wheat and TaDOF7.6-CRISPR/Cas9 gene-edited wheat previously constructed in our laboratory were used as experimental materials to evaluate their yield-related traits, molecular profiles and partial biosafety indicators. RESULTS: Both RNAi-mediated silencing and CRISPR/Cas9-mediated knockout of TaDOF7.6 significantly improved wheat grain traits and yield-related agronomic traits, further confirming that TaDOF7.6 acts as a negative regulator in wheat yield regulation. For TaDOF7.6-RNAi transgenic wheat, the integration site of T-DNA on wheat chromosome 3B was identified via thermal asymmetric interlaced PCR (TAIL-PCR). Vector-derived fragments including Hyg, GUS and dsRNA were consistently detectable across T3 to T5 generations. Moreover, these RNAi lines maintained stable histochemical GUS activity and sustained silencing efficiency of TaDOF7.6. No significant differences were observed in seed germination, seedling emergence and pollen-related traits between RNAi-25, RNAi-31 lines and wild-type wheat. Within the sampling scope and conventional PCR detection conditions adopted in this research, no target exogenous gene fragments were detected in 24 surrounding plant species. Additionally, there were no obvious disparities in field insect community and weed distribution between transgenic lines and wild-type controls. For TaDOF7.6-CRISPR/Cas9 gene-edited wheat, the insertion locus of exogenous vector on wheat chromosome 4D was successfully mapped. Transgene-free homozygous mutants were acquired through Hi-TOM high-throughput sequencing, Sanger sequencing and progeny screening. No unintended sequence variations were detected at all predicted off-target sites compared with wild-type plants. Meanwhile, the gene-edited lines exhibited consistent performance with wild-type wheat in seed germination, seedling emergence and pollen-associated phenotypes. CONCLUSION: This study provides fundamental data for molecular characterization and comprehensive biosafety assessment of TaDOF7.6-RNAi transgenic wheat and TaDOF7.6-CRISPR/Cas9 gene-edited wheat, and verifies the application potential of TaDOF7.6 as a promising target gene for wheat yield improvement. The transgene-free gene-edited mutants obtained in this work also supply elite new germplasm resources for subsequent wheat molecular breeding programs.
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US defence agencies grapple with gene drives.
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Tetrazine-Ligated CRISPR sgRNAs for Efficient Genome Editing.
CRISPR-Cas technology has revolutionized genome editing. Its broad and fast-growing application in biomedical research and therapeutics has led to increased demand for guide RNAs. The synthesis of chemically modified single-guide RNAs (sgRNAs) containing >100 nucleotides remains a bottleneck. Here we report the development of a tetrazine ligation method for the preparation of sgRNAs. A tetrazine moiety on the 3'-end of the crRNA and a norbornene moiety on the 5'-end of the tracrRNA enable successful ligation between crRNA and tracrRNA to form sgRNA under mild conditions. Tetrazine-ligated sgRNAs allow efficient genome editing of reporter and endogenous loci in human cells. High-efficiency editing requires structural optimization of the linker.
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TALEN outperforms Cas9 in editing heterochromatin target sites.
Genome editing critically relies on selective recognition of target sites. However, despite recent progress, the underlying search mechanism of genome-editing proteins is not fully understood in the context of cellular chromatin environments. Here, we use single-molecule imaging in live cells to directly study the behavior of CRISPR/Cas9 and TALEN. Our single-molecule imaging of genome-editing proteins reveals that Cas9 is less efficient in heterochromatin than TALEN because Cas9 becomes encumbered by local searches on non-specific sites in these regions. We find up to a fivefold increase in editing efficiency for TALEN compared to Cas9 in heterochromatin regions. Overall, our results show that Cas9 and TALEN use a combination of 3-D and local searches to identify target sites, and the nanoscopic granularity of local search determines the editing outcomes of the genome-editing proteins. Taken together, our results suggest that TALEN is a more efficient gene-editing tool than Cas9 for applications in heterochromatin.
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Plasmid-based complementation of large deletions in Phaeodactylum tricornutum biosynthetic genes generated by Cas9 editing.
The model diatom Phaeodactylum tricornutum is an attractive candidate for synthetic biology applications. Development of auxotrophic strains of P. tricornutum would provide alternative selective markers to commonly used antibiotic resistance genes. Here, using CRISPR/Cas9, we show successful editing of genes in the uracil, histidine, and tryptophan biosynthetic pathways. Nanopore long-read sequencing indicates that editing events are characterized by the occurrence of large deletions of up to ~ 2.7 kb centered on the editing site. The uracil and histidine-requiring phenotypes can be complemented by plasmid-based copies of the intact genes after curing of the Cas9-editing plasmid. Growth of uracil auxotrophs on media supplemented with 5-fluoroorotic acid and uracil results in loss of the complementing plasmid, providing a facile method for plasmid curing with potential applications in strain engineering and CRISPR editing. Metabolomic characterization of uracil auxotrophs revealed changes in cellular orotate concentrations consistent with partial or complete loss of orotate phosphoribosyltransferase activity. Our results expand the range of P. tricornutum auxotrophic strains and demonstrate that auxotrophic complementation markers provide a viable alternative to traditionally used antibiotic selection markers. Plasmid-based auxotrophic markers should expand the range of genome engineering applications and provide a means for biocontainment of engineered P. tricornutum strains.
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Functionalized PDA/DEX-PEI@HA nanoparticles combined with sleeping-beauty transposons for multistage targeted delivery of CRISPR/Cas9 gene.
CRISPR/Cas9 system has been used as the most powerful gene editing tool for precision medicine and advanced gene therapy. However, its wide applications are limited by the poor biosafety of lentivirus delivery vectors though with high-efficiency transduction. To construct a safer vector and promote genome integration, the CRISPR/Cas9 gene is cloned into a plasmid-based non-viral safe vector Sleeping-Beauty (SB) transposon in this study to obtain pT2SpCas9. Meanwhile, PDA/DEX-PEI@HA (PDPH) nanoparticles are constructed to facilitate the precise CRISPR/Cas9 targeting delivery, by using polydopamine (PDA) as the carrier, hyaluronic acid (HA) as the cell-targeting ligand and dexamethasone (DEX) as the nuclear localization signal (NLS). The results showed that PDPH could deliver pDNA efficiently into the cell and further into the nucleus. The transfection efficiency of PDPH is much higher than that of NPs without HA and DEX. Remarkably, the cytotoxicity of PDPH is negligible in comparison to PEI25k and PEI10k. Western blots showed that after the transfection of PDPH/pT2SpCas9-Nanog/SB11, Nanog protein in HeLa cells is knocked out, and the proliferation and migration abilities of tumor cells are significantly decreased. This study demonstrates that PDA/DEX-PEI25k@HA/pT2SpCas9 (PDPH25 K/pT2SpCas9) has the great potential as a non-viral gene vector for CRISPR/Cas9 delivery and clinical medication.
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Establishment of a DNA-free genome editing and protoplast regeneration method in cultivated tomato (Solanum lycopersicum).
We have established a DNA-free genome editing method via ribonucleoprotein-based CRISPR/Cas9 in cultivated tomato and obtained mutant plants regenerated from transfected protoplasts with a high mutation rate. The application of genome editing as a research and breeding method has provided many possibilities to improve traits in many crops in recent years. In cultivated tomato (Solanum lycopersicum), so far only stable Agrobacterium-mediated transformation carrying CRISPR/Cas9 reagents has been established. Shoot regeneration from transfected protoplasts is the major bottleneck in the application of DNA-free genome editing via ribonucleoprotein-based CRISPR/Cas9 method in cultivated tomato. In this study, we report the implementation of a transgene-free breeding method for cultivated tomato by CRISPR/Cas9 technology, including the optimization of protoplast isolation and overcoming the obstacle in shoot regeneration from transfected protoplasts. We have identified that the shoot regeneration medium containing 0.1 mg/L IAA and 0.75 mg/L zeatin was the best hormone combination with a regeneration rate of up to 21.3%. We have successfully obtained regenerated plants with a high mutation rate four months after protoplast isolation and transfection. Out of 110 regenerated M0 plants obtained, 35 (31.8%) were mutated targeting both SP and SP5G genes simultaneously and the editing efficiency was up to 60% in at least one allele in either SP or SP5G genes.
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