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Methods and advances in RNA characterization and design.
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Efficient Generation of Endogenous Fluorescent Reporters by Nested CRISPR in Caenorhabditis elegans.
CRISPR-based genome-editing methods in model organisms are evolving at an extraordinary speed. Whereas the generation of deletion or missense mutants is quite straightforward, the production of endogenous fluorescent reporters is more challenging. We have developed Nested CRISPR, a cloning-free ribonucleoprotein-driven method that robustly produces endogenous fluorescent reporters with EGFP, mCherry or wrmScarlet in Caenorhabditis elegans This method is based on the division of the fluorescent protein (FP) sequence in three fragments. In the first step, single-stranded DNA (ssDNA) donors (≤200 bp) are used to insert the 5' and 3' fragments of the FP in the locus of interest. In the second step, these sequences act as homology regions for homology-directed repair using a double-stranded DNA (dsDNA) donor (PCR product) containing the middle fragment, thus completing the FP sequence. In Nested CRISPR, the first step involving ssDNA donors is a well-established method that yields high editing efficiencies, and the second step is reliable because it uses universal CRISPR RNAs (crRNAs) and PCR products. We have also used Nested CRISPR in a nonessential gene to produce a deletion mutant in the first step and a transcriptional reporter in the second step. In the search for modifications to optimize the method, we tested synthetic single guide RNAs (sgRNAs), but did not observe a significant increase in efficiency. To streamline the approach, we combined all step 1 and step 2 reagents in a single injection and were successful in three of five loci tested with editing efficiencies of up to 20%. Finally, we discuss the prospects of this method in the future.
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Protocol for assessment of the efficiency of CRISPR/Cas RNP delivery to different types of target cells.
BACKGROUND: Delivery of CRISPR/Cas RNPs to target cells still remains the biggest bottleneck to genome editing. Many efforts are made to develop efficient CRISPR/Cas RNP delivery methods that will not affect viability of target cell dramatically. Popular current methods and protocols of CRISPR/Cas RNP delivery include lipofection and electroporation, transduction by osmocytosis and reversible permeabilization and erythrocyte-based methods. METHODS: In this study we will assess the efficiency and optimize current CRISPR/Cas RNP delivery protocols to target cells. We will conduct our work using molecular cloning, protein expression and purification, cell culture, flow cytometry (immunocytochemistry) and cellular imaging techniques. DISCUSSION: This will be the first extensive comparative study of popular current methods and protocols of CRISPR/Cas RNP delivery to human cell lines and primary cells. All protocols will be optimized and characterized using the following criteria i) protein delivery and genome editing efficacy; ii) viability of target cells after delivery (post-transduction recovery); iii) scalability of delivery process; iv) cost-effectiveness of the delivery process and v) intellectual property rights. Some methods will be considered 'research-use only', others will be recommended for scaling and application in the development of cell-based therapies.
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Customizable virus-like particles deliver CRISPR-Cas9 ribonucleoprotein for effective ocular neovascular and Huntington's disease gene therapy.
In vivo CRISPR gene editing holds enormous potential for various diseases. Ideally, CRISPR delivery should be cell type-specific and time-restricted for optimal efficacy and safety, but customizable methods are lacking. Here we develop a cell-tropism programmable CRISPR-Cas9 ribonucleoprotein delivery system (RIDE) based on virus-like particles. The efficiency of RIDE was comparable to that of adeno-associated virus and lentiviral vectors and higher than lipid nanoparticles. RIDE could be readily reprogrammed to target dendritic cells, T cells and neurons, and significantly ameliorated the disease symptoms in both ocular neovascular and Huntington's disease models via cell-specific gene editing. In addition, RIDE could efficiently edit the huntingtin gene in patients' induced pluripotent stem cell-derived neurons and was tolerated in non-human primates. This study is expected to facilitate the development of in vivo CRISPR therapeutics.
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Gene editing strategies to treat lysosomal disorders: The example of mucopolysaccharidoses.
Lysosomal storage disorders are a group of progressive multisystemic hereditary diseases with a combined incidence of 1:4,800. Here we review the clinical and molecular characteristics of these diseases, with a special focus on Mucopolysaccharidoses, caused primarily by the lysosomal storage of glycosaminoglycans. Different gene editing techniques can be used to ameliorate their symptoms, using both viral and nonviral delivery methods. Whereas these are still being tested in animal models, early results of phase I/II clinical trials of gene therapy show how this technology may impact the future treatment of these diseases. Hurdles related to specific hard-to-reach organs, such as the central nervous system, heart, joints, and the eye must be tackled. Finally, the regulatory framework necessary to advance into clinical practice is also discussed.
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Genome Editing by CRISPR/Cas12 Recognizing AT-Rich PAMs in Shewanella oneidensis MR-1.
Homologous recombination-mediated genomic editing is urgently needed to obtain high-performance chassis of electroactive microorganisms. However, the existing tools cannot meet the requirement of genome-wide editing in Shewanella oneidensis. Here, we develop different CRISPR-Cas systems that are ideal to be employed in AT-rich sequences as the supplements to Cas9. AsCpf1 and BhCas12b show low cell toxicity and superior ability to target sequences and are thus screened out in S. oneidensis MR-1. The PAMs of AsCpf1 and BhCas12b are 5'-TTTV-3' and 5'-ATTN-3'. For gene deletion, ∼1-kb gene is knocked out and the editing efficiency is 41.67% by BhCas12b-mediated system. For gene replacement, endogenous promoter of nagK was substituted to a constitutive promoter with the efficiency of 25% through BhCas12b system. For gene insertion, the integration efficiency was up to 94.4% and 83.9% via CRISPR-BhCas12b and AsCpf1 tools. This study implies a great potential of CRISPR-BhCas12b/AsCpf1 systems recognizing AT-rich PAMs for genomic editing in S. oneidensis to facilitate multifaceted gene manipulation.
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Negative DNA supercoiling induces genome-wide Cas9 off-target activity.
CRISPR-Cas9 is a powerful gene-editing technology; however, off-target activity remains an important consideration for therapeutic applications. We have previously shown that force-stretching DNA induces off-target activity and hypothesized that distortions of the DNA topology in vivo, such as negative DNA supercoiling, could reduce Cas9 specificity. Using single-molecule optical-tweezers, we demonstrate that negative supercoiling λ-DNA induces sequence-specific Cas9 off-target binding at multiple sites, even at low forces. Using an adapted CIRCLE-seq approach, we detect over 10,000 negative-supercoiling-induced Cas9 off-target double-strand breaks genome-wide caused by increased mismatch tolerance. We further demonstrate in vivo that directed local DNA distortion increases off-target activity in cells and that induced off-target events can be detected during Cas9 genome editing. These data demonstrate that Cas9 off-target activity is regulated by DNA topology in vitro and in vivo, suggesting that cellular processes, such as transcription and replication, could induce off-target activity at previously overlooked sites.
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CRISPR/Cas9 and piggyBac Transposon-Based Conversion of a Pathogenic Biallelic TBCD Variant in a Patient-Derived iPSC Line Allows Correction of PEBAT-Related Endophenotypes.
Induced pluripotent stem cells (iPSCs) have been established as a reliable in vitro disease model system and represent a particularly informative tool when animal models are not available or do not recapitulate the human pathophenotype. The recognized limit in using this technology is linked to some degree of variability in the behavior of the individual patient-derived clones. The development of CRISPR/Cas9-based gene editing solves this drawback by obtaining isogenic iPSCs in which the genetic lesion is corrected, allowing a straightforward comparison with the parental patient-derived iPSC lines. Here, we report the generation of a footprint-free isogenic cell line of patient-derived TBCD-mutated iPSCs edited using the CRISPR/Cas9 and piggyBac technologies. The corrected iPSC line had no genetic footprint after the removal of the selection cassette and maintained its "stemness". The correction of the disease-causing TBCD missense substitution restored proper protein levels of the chaperone and mitotic spindle organization, as well as reduced cellular death, which were used as read-outs of the TBCD KO-related endophenotype. The generated line represents an informative in vitro model to understand the impact of pathogenic TBCD mutations on nervous system development and physiology.
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Improved split prime editors enable efficient in vivo genome editing.
Efficient prime editor (PE) delivery in vivo is critical for realizing its full potential in disease modeling and therapeutic correction. Although PE has been divided into two halves and delivered using dual adeno-associated viruses (AAVs), the editing efficiency at different gene loci varies among split sites. Furthermore, efficient split sites within Cas9 nickase (Cas9n) are limited. Here, we verified that 1115 (Asn) is an efficient split site when delivering PEs by dual AAVs. Additionally, we utilized a feature in which reverse transcriptase could be detached from the Cas9n and designed split sites in the first half of Cas9n. We found that split-PE-367 enabled high editing efficiency with Rma intein. To test the editing efficiency in vivo, split-ePE3-367 was packaged in AAV9 and achieved 17.5% precise editing in mice. Our findings establish an alternative split-PE architecture that enables robust editing efficiency, facilitating potential utility in disease modeling and correction.
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A transferrable and integrative type I-F Cascade for heterologous genome editing and transcription modulation.
The Class 1 type I CRISPR-Cas systems represent the most abundant and diverse CRISPR systems in nature. However, their applications for generic genome editing have been hindered due to difficulties of introducing the class-specific, multi-component effectors (Cascade) in heterologous hosts for functioning. Here we established a transferrable Cascade system that enables stable integration and expression of a highly active type I-F Cascade in heterologous bacterial hosts for various genetic exploitations. Using the genetically recalcitrant Pseudomonas species as a paradigm, we show that the transferred Cascade displayed substantially higher DNA interference activity and greater editing capacity than both the integrative and plasmid-borne Cas9 systems, and enabled deletion of large fragments such as the 21-kb integrated cassette with efficiency and simplicity. An advanced I-F-λred system was further developed to enable editing in genotypes with poor homologous recombination capacity, clinical isolates lacking sequence information, and cells containing anti-CRISPR elements Acrs. Lastly, an 'all-in-one' I-F Cascade-mediated CRISPRi platform was developed for transcription modulation by simultaneous introduction of the Cascade and the programmed mini-CRISPR array in one-step. This study provides a framework for expanding the diverse type I Cascades for widespread, heterologous genome editing and establishment of editing techniques in 'non-model' bacterial species.
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