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Phosphate Lock Residues of Acidothermus cellulolyticus Cas9 Are Critical to Its Substrate Specificity.
Despite being utilized widely in genome sciences, CRISPR-Cas9 remains limited in achieving high fidelity in cleaving DNA. A better understanding of the molecular basis of Cas9 holds the key to improve Cas9-based tools. We employed direct evolution and in vitro characterizations to explore structural parameters that impact the specificity of the thermophilic Cas9 from Acidothermus cellulolyticus (AceCas9). By identifying variants that are able to cleave mismatched protospacers within the seed region, we found a critical role of the phosphate lock residues in substrate specificity in a manner that depends on their sizes and charges. Removal of the negative charge from the phosphate lock residues significantly decreases sensitivity to the guide-DNA mismatches. An increase in size of the substituted residues further reduces the sensitivity to mismatches at the first position of the protospacer. Our findings identify the phosphate lock residues as an important site for tuning the specificity and catalytic efficiency of Cas9.
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PE-Designer and PE-Analyzer: web-based design and analysis tools for CRISPR prime editing.
Prime editing technology is capable of generating targeted insertions, deletions, and base conversions. However, the process of designing prime editing guide RNAs (pegRNAs), which contain a primer binding site and a reverse-transcription template at the 3' end, is more complex than that for the single guide RNAs used with CRISPR nucleases or base editors. Furthermore, the assessment of high-throughput sequencing data after prime editors (PEs) have been employed should consider the unique feature of PEs; thus, pre-existing assessment tools cannot directly be adopted for PEs. Here, we present two user-friendly web-based tools for PEs, named PE-Designer and PE-Analyzer. PE-Designer, a dedicated tool for pegRNA selection, provides all possible target sequences, pegRNA extension sequences, and nicking guide RNA sequences together with useful information, and displays the results in an interactive image. PE-Analyzer, a dedicated tool for PE outcome analysis, accepts high-throughput sequencing data, summarizes mutation-related information in a table, and provides interactive graphs. PE-Analyzer was mainly written using JavaScript so that it can analyze several data sets without requiring that huge sequencing data (>100MB) be uploaded to the server, reducing analysis time and increasing personal security. PE-Designer and PE-Analyzer are freely available at http://www.rgenome.net/pe-designer/ and http://www.rgenome.net/pe-analyzer/ without a login process.
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Rapid and Efficient Genome Editing in Staphylococcus aureus by Using an Engineered CRISPR/Cas9 System.
Staphylococcus aureus, a major human pathogen, has been the cause of serious infectious diseases with a high mortality rate. Although genetics is a key means to study S. aureus physiology, such as drug resistance and pathogenesis, genetic manipulation in S. aureus is always time-consuming and labor-intensive. Here we report a CRISPR/Cas9 system (pCasSA) for rapid and efficient genome editing, including gene deletion, insertion, and single-base substitution mutation in S. aureus. The designed pCasSA system is amenable to the assembly of spacers and repair arms by Golden Gate assembly and Gibson assembly, respectively, enabling rapid construction of the plasmids for editing. We further engineered the pCasSA system to be an efficient transcription inhibition system for gene knockdown and possible genome-wide screening. The development of the CRISPR/Cas9-mediated genome editing and transcription inhibition tools will dramatically accelerate drug-target exploration and drug development.
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A Single Administration of CRISPR/Cas9 Lipid Nanoparticles Achieves Robust and Persistent In Vivo Genome Editing.
The development of clinically viable delivery methods presents one of the greatest challenges in the therapeutic application of CRISPR/Cas9 mediated genome editing. Here, we report the development of a lipid nanoparticle (LNP)-mediated delivery system that, with a single administration, enabled significant editing of the mouse transthyretin (Ttr) gene in the liver, with a >97% reduction in serum protein levels that persisted for at least 12 months. These results were achieved with an LNP delivery system that was biodegradable and well tolerated. The LNP delivery system was combined with a sgRNA having a chemical modification pattern that was important for high levels of in vivo activity. The formulation was similarly effective in a rat model. Our work demonstrates that this LNP system can deliver CRISPR/Cas9 components to achieve clinically relevant levels of in vivo genome editing with a concomitant reduction of TTR serum protein, highlighting the potential of this system as an effective genome editing platform.
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Highly efficient site-specific integration of DNA fragments into the honeybee genome using CRISPR/Cas9.
Functional genetic studies in honeybees have been limited to transposon mediated transformation and site directed mutagenesis tools. However, site- and sequence-specific manipulations that insert DNA fragments or replace sequences at specific target sites are lacking. Such tools would enable the tagging of proteins, the expression of reporters and site-specific amino acid changes, which are all gold standard manipulations for physiological, organismal, and genetic studies. However, such manipulations must be very efficient in honeybees since screening and crossing procedures are laborious due to their social organization. Here, we report an accurate and remarkably efficient site-specific integration of DNA-sequences into the honeybee genome using clustered regularly interspaced short palindromic repeat/clustered regularly interspaced short palindromic repeat-associated protein 9-mediated homology-directed repair. We employed early embryonic injections and selected a highly efficient sgRNA in order to insert 294 and 729 bp long DNA sequences into a specific locus at the dsx gene. These sequences were locus-specifically integrated in 57% and 59% of injected bees. Most importantly, 21% and 25% of the individuals lacked the wildtype sequence demonstrating that we generated homozygous mutants in which all cells are affected (no mosaicism). The highly efficient, locus-specific insertions of nucleotide sequences generating homozygous mutants demonstrate that systematic molecular studies for honeybees are in hand that allow somatic mutation approaches via workers or studies in the next generation using queens with their worker progeny. The employment of early embryonic injections and screenings of highly efficient sgRNAs may offer the prospect of highly successful sequence- and locus-specific mutations also in other organisms.
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CRISPR/Cas9-based Targeted Genome Editing for the Development of Monogenic Diseases Models with Human Pluripotent Stem Cells.
Human pluripotent stem cells (hPSCs) represent a formidable tool for disease modeling, drug discovery, and regenerative medicine using human cells and tissues in vitro. Evolving techniques of targeted genome editing, specifically the CRISPR/Cas9 system, allow for the generation of cell lines bearing gene-specific knock-outs, knock-in reporters, and precise mutations. However, there are increasing concerns related to the transfection efficiency, cell viability, and maintenance of pluripotency provided by genome-editing techniques. The procedure presented here employs transient antibiotic selection that overcomes reduced transfection efficiency, avoids cytotoxic flow sorting for increased viability, and generates multiple genome-edited pluripotent hPSC lines expanded from a single parent cell. Avoidance of xenogeneic contamination from feeder cells and reduced operator workload, owing to single-cell passaging rather than clump passaging, are additional benefits. The outlined methods may enable researchers with limited means and technical experience to create human stem cell lines containing desired gene-specific mutations. © 2018 by John Wiley & Sons, Inc.
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CRISPR/Cas9 directed to the Ube3a antisense transcript improves Angelman syndrome phenotype in mice.
Gene editing holds the potential to correct mutations and cure devastating genetic disorders. The technology has not yet proven efficacious for therapeutic use in CNS diseases with ubiquitous neuronal defects. Angelman syndrome (AS), a severe neurodevelopmental disorder, is caused by a lack of maternal expression of the UBE3A gene. Because of genomic imprinting, only neurons are affected. One therapeutic approach focuses on the intact paternal UBE3A copy in patients with AS that is silenced by an antisense transcript (UBE3A-ATS). We show here that gene editing of Ube3a-ATS in the mouse brain resulted in the formation of base pair insertions/deletions (indels) in neurons and the subsequent unsilencing of the paternal Ube3a allele in neurons, which partially corrected the behavioral phenotype of a murine AS model. This study provides compelling evidence to further investigate editing of the homologous region of the human UBE3A-ATS because this may provide a lasting therapeutic effect for patients with AS.
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GFP-on mouse model for interrogation of in vivo gene editing.
Gene editing technologies have revolutionized therapies for numerous genetic diseases. However, in vivo gene editing hinges on identifying efficient delivery vehicles for editing in targeted cell types, a significant hurdle in fully realizing its therapeutic potential. A model system to rapidly evaluate systemic gene editing would advance the field. Here, we develop the GFP-on reporter mouse, which harbors a nonsense mutation in a genomic EGFP sequence correctable by adenine base editor (ABE) among other genome editors. The GFP-on system was validated using single and dual adeno-associated virus (AAV9) encoding ABE8e and sgRNA. Intravenous administration of AAV9-ABE8e-sgRNA into adult GFP-on mice results in EGFP expression consistent with the tropism of AAV9. Intrahepatic delivery of AAV9-ABE8e-sgRNA into GFP-on fetal mice restores EGFP expression in AAV9-targeted organs lasting at least six months post-treatment. The GFP-on model provides an ideal platform for high-throughput evaluation of emerging gene editing tools and delivery modalities.
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Novel therapies in β-thalassaemia.
Beta-thalassaemia is one of the most significant haemoglobinopathies worldwide resulting in the synthesis of little or no β-globin chains. Without treatment, β-thalassaemia major is lethal within the first decade of life due to the complex pathophysiology, which leads to wide clinical manifestations. Current clinical management for these patients depends on repeated transfusions followed by iron-chelating therapy. Several novel approaches to correct the resulting α/β-globin chain imbalance, treat ineffective erythropoiesis and improve iron overload are currently being developed. Up to now, the only curative treatment for β-thalassemia is haematopoietic stem-cell transplantation, but this is a risky and costly procedure. Gene therapy, gene editing and base editing are emerging as a powerful approach to treat this disease. In β-thalassaemia, gene therapy involves the insertion of a vector containing the normal β-globin or γ-globin gene into haematopoietic stem cells to permanently produce normal red blood cells. Gene editing and base editing involves the use of zinc finger nucleases, transcription activator-like nucleases and clustered regularly interspaced short palindromic repeats/Cas9 to either correct the causative mutation or else insert a single nucleotide variant that will increase foetal haemoglobin. In this review, we will examine the current management strategies used to treat β-thalassaemia and focus on the novel therapies targeting ineffective erythropoiesis, improving iron overload and correction of the globin chain imbalance.
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Identification and Analysis of Small Molecule Inhibitors of CRISPR-Cas9 in Human Cells.
Genome editing tools based on CRISPR-Cas systems can repair genetic mutations in situ; however, off-target effects and DNA damage lesions that result from genome editing remain major roadblocks to its full clinical implementation. Protein and chemical inhibitors of CRISPR-Cas systems may reduce off-target effects and DNA damage. Here we describe the identification of several lead chemical inhibitors that could specifically inhibit the activity of Streptococcus pyogenes Cas9 (SpCas9). In addition, we obtained derivatives of lead inhibitors that could penetrate the cell membrane and inhibit SpCas9 in cellulo. Two of these compounds, SP2 and SP24, were able to improve the specificity of SpCas9 in cellulo at low-micromolar concentration. Furthermore, microscale thermophoresis (MST) assays showed that SP24 might inhibit SpCas9 activity by interacting with both the SpCas9 protein and the SpCas9-gRNA ribonucleoprotein complex. Taken together, SP24 is a novel chemical inhibitor of SpCas9 which has the potential to enhance therapies that utilize SpCas9.
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