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Disrupting Protein Expression with Double-Clicked sgRNA-Cas9 Complexes: A Modular Approach to CRISPR Gene Editing.
CRISPR-Cas9 is currently the most versatile technique to perform gene editing in living organisms. In this approach, the Cas9 endonuclease is guided toward its DNA target sequence by the guide RNA (gRNA). Chemical synthesis of a functional single gRNA (sgRNA) is nontrivial because of the length of the RNA strand. Recently we demonstrated that a sgRNA can be stitched together from three smaller fragments through a copper-catalyzed azide-alkyne cycloaddition, making the process highly modular. Here we further advance this approach by leveraging this modulator platform by incorporating chemically modified nucleotides at both ends of the modular sgRNA to increase resistance against ribonucleases. Modified nucleotides consisted of a 2'-O-Me group and a phosphorothioate backbone in varying number at both the 5'- and 3'-ends of the sgRNA. It was observed that three modified nucleotides at both ends of the sgRNA significantly increased the success of Cas9 in knocking out a gene of interest. Using these chemically stabilized sgRNAs facilitates multigene editing at the protein level, as demonstrated by successful knockout of both Siglec-3 and Siglec-7 using two fluorophores in conjunction with fluorescence-activated cell sorting. These results demonstrate the versatility of this modular platform for assembling sgRNAs from small, chemically modified strands to simultaneously disrupt the gene expression of two proteins.
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Harnessing noncanonical crRNAs to improve functionality of Cas12a orthologs.
There is a broad diversity among Cas12a endonucleases that possess nucleic acid detection and gene-editing capabilities, but few are studied extensively. Here, we present an exhaustive investigation of 23 Cas12a orthologs, with a focus on their cis- and trans-cleavage activities in combination with noncanonical crRNAs. Through biochemical assays, we observe that some noncanonical crRNA:Cas12a effector complexes outperform their corresponding wild-type crRNA:Cas12a. Cas12a can recruit crRNA with modifications such as loop extensions and split scaffolds. Moreover, the tolerance of Cas12a to noncanonical crRNA is also observed in mammalian cells through the formation of indels. We apply the adaptability of Cas12a:crRNA complexes to detect SARS-CoV-2 in clinical nasopharyngeal swabs, saliva samples, and tracheal aspirates. Our findings further expand the toolbox for next-generation CRISPR-based diagnostics and gene editing.
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A simple protocol to isolate a single human cell PRDX1 knockout generated by CRISPR-Cas9 system.
Here, we describe a protocol for human PRDX1 gene knockout cells using the CRISPR-Cas9 system. The protocol describes all the steps sequentially: (1) single-guide RNA design, cloning, and transfection; (2) gene editing evaluation by T7EI assay; (3) single-cell isolation; and (4) knockout verification to determine indels in one or both alleles by Sanger sequencing. This strategy is based on the efficiency of DNA editing, avoids antibiotic selection, and bypasses the need for cell sorting.
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Clinical progress in genome-editing technology and in vivo delivery techniques.
There is wide interest in applying genome-editing tools to prevent, treat, and cure a variety of diseases. Since the discovery of the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) systems, these techniques have been used in combination with different delivery systems to create highly efficacious treatment options. Each delivery system has its own advantages and disadvantages and is being used for various applications. With the large number of gene-editing applications being studied but very few being brought into the clinic, we review current progress in the field, specifically where genome editing has been applied in vivo and in the clinic, and identify current challenges and areas of future growth.
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Past, present, and future of CRISPR genome editing technologies.
Genome editing has been a transformative force in the life sciences and human medicine, offering unprecedented opportunities to dissect complex biological processes and treat the underlying causes of many genetic diseases. CRISPR-based technologies, with their remarkable efficiency and easy programmability, stand at the forefront of this revolution. In this Review, we discuss the current state of CRISPR gene editing technologies in both research and therapy, highlighting limitations that constrain them and the technological innovations that have been developed in recent years to address them. Additionally, we examine and summarize the current landscape of gene editing applications in the context of human health and therapeutics. Finally, we outline potential future developments that could shape gene editing technologies and their applications in the coming years.
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When is a GM plant not a GM plant?
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Use of CRISPR/Cas ribonucleoproteins for high throughput gene editing of induced pluripotent stem cells.
Induced pluripotent stem cells (iPSCs) have become widely used for disease modelling, particularly with regard to predisposing genetic risk factors and causal gene variants. Alongside this, technologies such as the CRISPR/Cas system have been adapted to enable programmable gene editing in human cells. When combined, CRISPR/Cas gene editing of donor-specific iPSC to generate isogenic cell lines that differ only at specific gene variants provides a powerful model with which to investigate genetic variants associated with diseases affecting many organs, including the brain and eye. Here we describe our optimized protocol for using CRISPR/Cas ribonucleoproteins to edit disease causing gene variants in human iPSCs. We discuss design of crRNAs and homology-directed repair templates, assembly of CRISPR/Cas ribonucleoproteins, optimization of delivery via nucleofection, and strategies for single cell cloning, efficient clone cryopreservation and genotyping for identifying iPSC clones for further characterization.
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In vitro CRISPR-Cas9-mediated efficient Ad5 vector modification.
The CRISPR-Cas9 genome editing system has been widely used in multiple cells and organisms. Here we developed a CRISPR-Cas9 based in vitro large DNA vector editing system, using the Ad5-based vector as an example. We demonstrate use of this system to generate targeted mutations, in-frame gene deletion, and gene replacement. This in vitro CRISPR editing system exhibits high efficiency and accuracy. We believe this system can be applied in a variety of experimental settings.
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Introduction to Gene Editing and Manipulation Using CRISPR/Cas9 Technology.
Until very recently, the prospect of introducing mutations or exogenous DNA sequences at precise locations in the genomes of plants and animals was difficult, if not impossible. This rapidly changed with the demonstration that the type II CRISPR-Cas complex, a bacterial anti-viral surveillance system, could be engineered into a simple and robust platform for introducing double-stranded DNA breaks at nearly any position of plant and animal genomes. The prospect of efficiently creating tailored changes to a gene of interest is revolutionizing biomedical research, allowing exciting new questions to be asked. This overview introduces CRISPR-Cas technology as a tool for molecular biology and briefly discusses the advantages of this method over earlier techniques, as well as unique opportunities to create new avenues of research. © 2016 by John Wiley & Sons, Inc.
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[Advances of using CRISPR-Cas13a system for tumor diagnosis and treatment].
CRISPR-Cas systems are well known gene editing tools, among which CRISPR-Cas9 system targeting DNA is the most well developed. Compared with CRISPR-Cas9 system, CRISPR-C2c2/ Cas13a system derived from TYPE VI of CRISPR family that can target RNA has attracted increasingly intense investigations in recent years. The CRISPR-Cas13a system is featured by specific recognition and binding of single stranded RNA sequences, thus playing a role in non-specific cleavage of RNA. This feature could be potentially applied to detect free nucleic acid in tumors or peripheral blood as a diagnostic approach. Since Cas13a specifically targets RNA, it can directly edit mRNA transcripts of genomic DNA to achieve the downregulation of target proteins without involving DNA editing. Therefore, Cas13a system could be used in tumor treatment. This review summarized the advances of using CRISPR-Cas13a for RNA targeting in tumor diagnosis and treatment, and prospected future applications.
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