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Cas9-mediated gene-editing frequency in microalgae is doubled by harnessing the interaction between importin α and phytopathogenic NLSs.
Pathogen-derived nuclear localization signals (NLSs) enable vigorous nuclear invasion in the host by the virulence proteins harboring them. Herein, inspired by the robust nuclear import mechanism, we show that NLSs originating from the plant infection-associated Agrobacterium proteins VirD2 and VirE2 can be incorporated into the Cas9 system as efficient nuclear delivery enhancers, thereby improving the low gene-editing frequency in a model microalga, Chlamydomonas reinhardtii, caused by poor nuclear localization of the bulky nuclease. Prior to evaluation of the NLSs, IPA1 (Cre04.g215850) was first defined in the alga as the nuclear import-related importin alpha (Impα) that serves as a counterpart adaptor protein of the NLSs, based on extensive in silico analyses considering the protein's sequence, tertiary folding behavior, and structural basis when interacting with a well-studied SV40TAg NLS. Through precursive affinity explorations, we reproducibly found that the NLSs mediated the binding between the Cas9 and Impα with nM affinities and visually confirmed that the fusion of the NLSs strictly localized the peptide-bearing cargoes in the microalgal nucleus without compensating for their cleavage ability. When employed in a real-world application, the VirD2 NLS increases the mutation frequency (~1.12 × 10-5) over 2.4-fold compared to an archetypal SV40TAg NLS (~0.46 × 10-5) when fused with Cas9. We demonstrate the cross-species versatility of the Impα-dependent strategy by successfully applying it to an industrial alga, Chlorella Sp. HS2. This work, focused on affinity augmentation, provides insights into increasing the frequency of gene editing, which can be advantageously used in programmable mutagenesis with broad applicability.
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A Simple and Efficient Protocol to Transform and Regenerate CRISPR-Cas9-Mediated Genome-Edited Tomato Plants.
CRISPR-Cas9-mediated genome editing has revolutionized functional genomics and crop improvement. However, to maximize the adoption of the CRISPR-Cas9 technology, an efficient method to transform and regenerate genetically edited plants is necessary. In this protocol, we describe a detailed method to generate a CRISPR-Cas9 construct based on the Golden Gate cloning system and a simple and efficient method to transform and regenerate tomato plants from cotyledons co-cultured with Agrobacterium. Our protocol allows the production of at least ten Cas-positive independent lines from one hundred cotyledons. This protocol is routinely used in our laboratory to obtain tomato mutant lines and has been proven effective across several genotypes.
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In Vivo CRISPR/Cas9 Gene Editing Corrects Retinal Dystrophy in the S334ter-3 Rat Model of Autosomal Dominant Retinitis Pigmentosa.
Reliable genome editing via Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 may provide a means to correct inherited diseases in patients. As proof of principle, we show that CRISPR/Cas9 can be used in vivo to selectively ablate the rhodopsin gene carrying the dominant S334ter mutation (Rho(S334)) in rats that model severe autosomal dominant retinitis pigmentosa. A single subretinal injection of guide RNA/Cas9 plasmid in combination with electroporation generated allele-specific disruption of Rho(S334), which prevented retinal degeneration and improved visual function.
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Improved CRISPR genome editing using small highly active and specific engineered RNA-guided nucleases.
Streptococcus pyogenes (Spy) Cas9 has potential as a component of gene therapeutics for incurable diseases. One of its limitations is its large size, which impedes its formulation and delivery in therapeutic applications. Smaller Cas9s are an alternative, but lack robust activity or specificity and frequently recognize longer PAMs. Here, we investigated four uncharacterized, smaller Cas9s and found three employing a "GG" dinucleotide PAM similar to SpyCas9. Protein engineering generated synthetic RNA-guided nucleases (sRGNs) with editing efficiencies and specificities exceeding even SpyCas9 in vitro and in human cell lines on disease-relevant targets. sRGN mRNA lipid nanoparticles displayed manufacturing advantages and high in vivo editing efficiency in the mouse liver. Finally, sRGNs, but not SpyCas9, could be packaged into all-in-one AAV particles with a gRNA and effected robust in vivo editing of non-human primate (NHP) retina photoreceptors. Human gene therapy efforts are expected to benefit from these improved alternatives to existing CRISPR nucleases.
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CRISPR/Cas9-Mediated Genome Editing System in Insect Genomics and Pest Management.
The CRISPR/Cas9 is being developed as an invaluable system that allows rapid and site-specific genome editing in a wide variety of organisms, including diverse insects. It has been successfully used for gene function annotations of RNAi pathway in insect genomics and will facilitate research on RNAi mechanism. Here, we describe a streamlined method to generate and detect somatic and germline knockout mutations of desired target genes in tephritid pests by injecting mRNA encoding the Cas9 endonuclease and in vitro transcribed single guide RNA (sgRNA) into embryos. Target site selection, sgRNA synthesis, Cas9 synthesis, microinjection, and mutation identification are presented in detail.
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Immunodeficiency: Gene therapy for primary immune deficiency.
Current gene therapy for inborn errors of immunity have involved the use of gene addition approaches with viral delivery. This main strategy has had demonstrated success mainly in severe combined immune deficiency, Wiskott-Aldrich syndrome, and chronic granulomatous disease. Despite the increasing success of gene therapy, there are limitations of gene therapy, and, therefore, hematopoietic stem cell transplantation continues to be the preferred option. With improvements in viral delivery through next-generation lentiviral vectors and the advent of gene editing with CRISPR-Cas9, the efficacy and safety of gene therapy may soon surpass hematopoietic stem cell transplantation. Furthermore, these advances improve the viability of gene therapy for inborn errors of immunity primarily through decreased risk of transplantation-related complications. Therefore, despite current limitations, gene therapy for inborn errors of immunity is poised to continue to expand to more patients and indications.
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Safety and efficacy of CRISPR-mediated genome ablation of VEGFA as a treatment for choroidal neovascularization in nonhuman primate eyes.
CRISPR-based genome editing enables permanent suppression of angiogenic factors such as vascular endothelial growth factor (VEGF) as a potential treatment for choroidal neovascularization (CNV)-a major cause of blindness in age-related macular degeneration. We previously designed adeno-associated viral (AAV) vectors with S. pyogenes Cas 9 (SpCas9) and guide RNAs (gRNAs) to target conserved sequences in VEGFA across mouse, rhesus macaque, and human, with successful suppression of VEGF and laser-induced CNV in mice. Here, we advanced the platform to nonhuman primates and found that subretinal AAV8-SpCas9 with gRNAs targeting VEGFA may reduce VEGF and CNV severity as compared with SpCas9 without gRNAs. However, all eyes that received AAV8-SpCas9 regardless of gRNA presence developed subfoveal deposits, concentric macular rings, and outer retinal disruption that worsened at higher dose. Immunohistochemistry showed subfoveal accumulation of retinal pigment epithelial cells, collagen, and vimentin, disrupted photoreceptor structure, and retinal glial and microglial activation. Subretinal AAV8-SpCas9 triggered aqueous elevations in CCL2, but minimal systemic humoral or cellular responses against AAV8, SpCas9, or GFP reporter. Our findings suggest that CRISPR-mediated VEGFA ablation in nonhuman primate eyes may suppress VEGF and CNV, but can also lead to unexpected subretinal fibrosis, photoreceptor damage, and retinal inflammation despite minimal systemic immune responses.
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Stimulation of homologous recombination in plants expressing heterologous recombinases.
BACKGROUND: Current excitement about the opportunities for gene editing in plants have been prompted by advances in CRISPR/Cas and TALEN technologies. CRISPR/Cas is widely used to knock-out or modify genes by inducing targeted double-strand breaks (DSBs) which are repaired predominantly by error-prone non-homologous end-joining or microhomology-mediated end joining resulting in mutations that may alter or abolish gene function. Although such mutations are random, they occur at sufficient frequency to allow useful mutations to be routinely identified by screening. By contrast, gene knock-ins to replace entire genes with alternative alleles or copies with specific characterised modifications, is not yet routinely possible. Gene replacement (or gene targeting) by homology directed repair occurs at extremely low frequency in higher plants making screening for useful events unfeasible. Homology directed repair might be increased by inhibiting non-homologous end-joining and/or stimulating homologous recombination (HR). Here we pave the way to increasing gene replacement efficiency by evaluating the effect of expression of multiple heterologous recombinases on intrachromosomal homologous recombination (ICR) in Nicotiana tabacum plants. RESULTS: We expressed several bacterial and human recombinases in different combinations in a tobacco transgenic line containing a highly sensitive β-glucuronidase (GUS)-based ICR substrate. Coordinated simultaneous expression of multiple recombinases was achieved using the viral 2A translational recoding system. We found that most recombinases increased ICR dramatically in pollen, where HR will be facilitated by the programmed DSBs that occur during meiosis. DMC1 expression produced the greatest stimulation of ICR in primary transformants, with one plant showing a 1000-fold increase in ICR frequency. Evaluation of ICR in homozygous T2 plant lines revealed increases in ICR of between 2-fold and 380-fold depending on recombinase(s) expressed. By comparison, ICR was only moderately increased in vegetative tissues and constitutive expression of heterologous recombinases also reduced plant fertility. CONCLUSION: Expression of heterologous recombinases can greatly increase the frequency of HR in plant reproductive tissues. Combining such recombinase expression with the use of CRISPR/Cas9 to induce DSBs could be a route to radically improving gene replacement efficiency in plants.
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Efficient and transformation-free genome editing in pepper enabled by RNA virus-mediated delivery of CRISPR/Cas9.
Tomato spotted wilt virus-mediated delivery of CRISPR/Cas9 bypasses the need for stable transformation and permits efficient, DNA-free genome editing in pepper. Remarkably, up to 77.9% of regenerated pepper plants contained heritable edits. This method has been validated with two pepper varieties and is compatible with existing tissue culture protocols.
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CRISPR Genome Editing Systems in the Genus Clostridium: a Timely Advancement.
The genus Clostridium is composed of bioproducers, which are important for the industrial production of chemicals, as well as pathogens, which are a significant burden to the patients and on the health care industry. Historically, even though these bacteria are well known and are commonly studied, the genetic technologies to advance our understanding of these microbes have lagged behind other systems. New tools would continue the advancement of our understanding of clostridial physiology. The genetic modification systems available in several clostridia are not as refined as in other organisms and each exhibit their own drawbacks. With the advent of the repurposing of the CRISPR-Cas systems for genetic modification, the tools available for clostridia have improved significantly over the past four years. Several CRISPR-Cas systems such as using wild-type Cas9, Cas9n, dCas9/CRISPR interference (CRISPRi) and a newly studied Cpf1/Cas12a, are reported. These have the potential to greatly advance the study of clostridial species leading to future therapies or the enhanced production of industrially relevant compounds. Here we discuss the details of the CRISPR-Cas systems as well as the advances and current issues in the developed clostridial systems.
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