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CRISPR-Cas9 Genome Editing of Rat Embryos using Adeno-Associated Virus (AAV) and 2-Cell Embryo Electroporation.
Genome editing technology is widely used to produce genetically modified animals, including rats. Cytoplasmic or pronuclear injection of DNA repair templates and CRISPR-Cas reagents is the most common delivery method into embryos. However, this type of micromanipulation necessitates access to specialized equipment, is laborious, and requires a certain level of technical skill. Moreover, microinjection techniques often result in lower embryo survival due to the mechanical stress on the embryo. In this protocol, we developed an optimized method to deliver large DNA repair templates to work in conjunction with CRISPR-Cas9 genome editing without the need for microinjection. This protocol combines AAV-mediated DNA delivery of single-stranded DNA donor templates along with the delivery of CRISPR-Cas9 ribonucleoprotein (RNP) by electroporation to modify 2-cell embryos. Using this novel strategy, we have successfully produced targeted knock-in rat models carrying insertion of DNA sequences from 1.2 to 3.0 kb in size with efficiencies between 42% and 90%.
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Simplifying protein engineering with deep learning.
When it comes to deep learning for protein engineering, there is strength in simplicity. In this issue of Cell, through thoughtful deployment of existing fixed-backbone sequence design models, Caixia Gao and colleagues engineer diverse genome editing systems with improved functionality, enabling powerful capabilities in fine-grained and large-scale genome editing as demonstrated through strong experimental validation.
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CRISPR-Cas: more than ten years and still full of mysteries.
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A Novel CRISPR-Cas9 Strategy to Target DYSTROPHIN Mutations Downstream of Exon 44 in Patient-Specific DMD iPSCs.
Mutations in the DMD gene cause fatal Duchenne Muscular Dystrophy (DMD). An attractive therapeutic approach is autologous cell transplantation utilizing myogenic progenitors derived from induced pluripotent stem cells (iPSCs). Given that a significant number of DMD mutations occur between exons 45 and 55, we developed a gene knock-in approach to correct any mutations downstream of exon 44. We applied this approach to two DMD patient-specific iPSC lines carrying mutations in exons 45 and 51 and confirmed mini-DYSTROPHIN (mini-DYS) protein expression in corrected myotubes by western blot and immunofluorescence staining. Transplantation of gene-edited DMD iPSC-derived myogenic progenitors into NSG/mdx4Cv mice produced donor-derived myofibers, as shown by the dual expression of human DYSTROPHIN and LAMIN A/C. These findings further provide proof-of-concept for the use of programmable nucleases for the development of autologous iPSC-based therapy for muscular dystrophies.
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Magnetically Driven High-Speed Rolling Nanoclusters for Enhanced CRISPR/Cas9 Genome Editing.
Despite the tremendous potential of the CRISPR/Cas9 gene-editing technology in precision therapeutics, intracellular delivery remains a major challenge. High cytoplasmic viscosity and lysosomal entrapment significantly impair the cytosolic transport and gene-editing efficiency. In this study, we demonstrate that both the size and magnetic responsiveness of Fe3O4 nanoclusters can be finely tuned by modulating ionic strength, enabling their rapid propulsion under external magnetic fields. Leveraging this property, we develop magnetic nanoparticle cluster nanorobots (MagCbots) of approximately 200 nm in size by electrostatically assembling Fe3O4 nanoclusters with CRISPR-Cas9 plasmids. Under magnetic actuation, MagCbots exhibit rapid rotation in highly viscous intracellular environments, achieving a linear velocity of ∼0.41 μm/s. MagCbots reduce intracellular viscosity by approximately 50% and enhance lysosomal escape efficiency by 3-fold compared to nonactuated counterparts. Their porous architecture not only offers high payload capacity but also protects plasmid DNA from enzymatic degradation. Notably, MagCbots enable efficient genome editing of both PD1 and PLK1 genes across various cell lines including hard-to-transfect Jurkat T cells. This magnetically driven nanorobot platform presents a promising strategy for active intracellular delivery and holds significant potential for advancing gene therapy and related biomedical applications.
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Digital droplet PCR and IDAA for the detection of CRISPR indel edits in the malaria species Anopheles stephensi.
CRISPR/Cas9 technology is a powerful tool for the design of gene-drive systems to control and/or modify mosquito vector populations; however, CRISPR/Cas9-mediated nonhomologous end joining mutations can have an important impact on generating alleles resistant to the drive and thus on drive efficiency. We demonstrate and compare the insertions or deletions (indels) detection capabilities of two techniques in the malaria vector mosquito Anopheles stephensi: Indel Detection by Amplicon Analysis (IDAA™) and Droplet Digital™ PCR (ddPCR™). Both techniques showed accuracy and reproducibility for indel frequencies across mosquito samples containing different ratios of indels of various sizes. Moreover, these techniques have advantages that make them potentially better suited for high-throughput nonhomologous end joining analysis in cage trials and contained field testing of gene-drive mosquitoes.
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CRISPR-Cas9 for the genome engineering of cyanobacteria and succinate production.
Cyanobacteria hold promise as a cell factory for producing biofuels and bio-derived chemicals, but genome engineering of cyanobacteria such as Synechococcus elongatus PCC 7942 poses challenges because of their oligoploidy nature and long-term instability of the introduced gene. CRISPR-Cas9 is a newly developed RNA-guided genome editing system, yet its application for cyanobacteria engineering has yet to be reported. Here we demonstrated that CRISPR-Cas9 system can effectively trigger programmable double strand break (DSB) at the chromosome of PCC 7942 and provoke cell death. With the co-transformation of template plasmid harboring the gene cassette and flanking homology arms, CRISPR-Cas9-mediated DSB enabled precise gene integration, ameliorated the homologous recombination efficiency and allowed the use of lower amount of template DNA and shorter homology arms. The CRISPR-Cas9-induced cell death imposed selective pressure and enhanced the chance of concomitant integration of gene cassettes into all chromosomes of PCC 7942, hence accelerating the process of obtaining homogeneous and stable recombinant strains. We further explored the feasibility of engineering cyanobacteria by CRISPR-Cas9-assisted simultaneous glgc knock-out and gltA/ppc knock-in, which improved the succinate titer to 435.0±35.0μg/L, an ≈11-fold increase when compared with that of the wild-type cells. These data altogether justify the use of CRISPR-Cas9 for genome engineering and manipulation of metabolic pathways in cyanobacteria.
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Advances in CRISPR-Cas9 in lineage tracing of model animals.
Cell lineage tracing is a key technology for describing the developmental history of individual progenitor cells and assembling them to form a lineage development tree. However, traditional methods have limitations of poor stability and insufficient resolution. As an efficient and flexible gene editing tool, CRISPR-Cas9 system has been widely used in biological research. Furthermore, CRISPR-Cas9 gene editing-based tracing methods can introduce fluorescent proteins, reporter genes, or DNA barcodes for high-throughput sequencing, enabling precise lineage analysis, significantly improving precision and resolution, and expanding its application range. In this review, we summarize applications of CRISPR-Cas9 system in cell lineage tracing, with special emphasis on its successful applications in traditional model animals (e.g., zebrafish and mice), large animal models (pigs), and human cells or organoids. We also discussed its potential prospects and challenges in xenotransplantation and regenerative medicine.
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Successful production of genome-edited rats by the rGONAD method.
BACKGROUND: Recent progress in development of the CRISPR/Cas9 system has been shown to be an efficient gene-editing technology in various organisms. We recently developed a novel method called Genome-editing via Oviductal Nucleic Acids Delivery (GONAD) in mice; a novel in vivo genome editing system that does not require ex vivo handling of embryos, and this technology is newly developed and renamed as "improved GONAD" (i-GONAD). However, this technology has been limited only to mice. Therefore in this study, we challenge to apply this technology to rats. RESULTS: Here, we determine the most suitable condition for in vivo gene delivery towards rat preimplantation embryos using tetramethylrhodamine-labelled dextran, termed as Rat improved GONAD (rGONAD). Then, to investigate whether this method is feasible to generate genome-edited rats by delivery of CRISPR/Cas9 components, the tyrosinase (Tyr) gene was used as a target. Some pups showed albino-colored coat, indicating disruption of wild-type Tyr gene allele. Furthermore, we confirm that rGONAD method can be used to introduce genetic changes in rat genome by the ssODN-based knock-in. CONCLUSIONS: We first establish the rGONAD method for generating genome-edited rats. We demonstrate high efficiency of the rGONAD method to produce knock-out and knock-in rats, which will facilitate the production of rat genome engineering experiment. The rGONAD method can also be readily applicable in mammals such as guinea pig, hamster, cow, pig, and other mammals.
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Targeted genome editing of the non-model cyanobacterium Cyanothece PCC 7425 via CRISPR/Cas12a.
Cyanobacteria are diverse photosynthetic microorganisms of great interest for fundamental science and sustainable biotechnological applications. However, their polyploidy makes genetic manipulation challenging and time-consuming. The development of CRISPR/Cas tools has greatly accelerated genome editing and metabolic engineering of some cyanobacterial model species. In this work, we extend the CRISPR/Cas12a system for targeted gene deletion in the non-model cyanobacterium Cyanothece sp. PCC 7425, interesting for its ability to perform intracellular calcium carbonate (CaCO3) biomineralization, nitrogen fixation, etc. We demonstrate for the first time its tractability to gene knockout by generating deletion mutants of four genes (cax3-cax4, gor, and sodB) acting in metabolism and/or response to stresses, using Cas12a-mediated homologous recombination. Importantly, full chromosome segregation was rapidly achieved after a single round of selection in all cases. All mutants were genotypically and phenotypically characterised. Moreover, biochemical analysis in the case of the ΔsodB mutant further confirmed its targeted deletion. Overall, CRISPR/Cas12a provides a rapid and efficient system for genome editing in Cyanothece sp. PCC 7425, establishing this organism as a versatile model for studying oxidative stress pathways, metal toxicity, and moreover, the still poorly known mechanism(s) of intracellular CaCO3 biomineralization. KEY POINTS: • Rapid and efficient CRISPR/Cas12a editing established in Cyanothece sp. PCC 7425. • Fully segregated knockout mutants obtained after a single selection round. • Platform for exploring the biotechnological potential of Cyanothece sp. PCC 7425.
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