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CRISPR-Based Precision Molecular Diagnostics for Disease Detection and Surveillance.
Sensitive, rapid, and portable molecular diagnostics is the future of disease surveillance, containment, and therapy. The recent SARS-CoV-2 pandemic has reminded us of the vulnerability of lives from ever-evolving pathogens. At the same time, it has provided opportunities to bridge the gap by translating basic molecular biology into therapeutic tools. One such molecular biology technique is CRISPR (clustered regularly interspaced short palindromic repeat) which has revolutionized the field of molecular diagnostics at the need of the hour. The use of CRISPR-Cas systems has been widespread in biology research due to the ease of performing genetic manipulations. In 2012, CRISPR-Cas systems were, for the first time, shown to be reprogrammable, i.e., capable of performing sequence-specific gene editing. This discovery catapulted the field of CRISPR-Cas research and opened many unexplored avenues in the field of gene editing, from basic research to therapeutics. One such field that benefitted greatly from this discovery was molecular diagnostics, as using CRISPR-Cas technologies enabled existing diagnostic methods to become more sensitive, accurate, and portable, a necessity in disease control. This Review aims to capture some of the trajectories and advances made in this arena and provides a comprehensive understanding of the methods and their potential use as point-of-care diagnostics.
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Guide RNAs: A Glimpse at the Sequences that Drive CRISPR-Cas Systems.
CRISPR-Cas systems provide adaptive immunity in bacteria and archaea. Although there are two main classes of CRISPR-Cas systems defined by gene content, interfering RNA biogenesis, and effector proteins, Type II systems have recently been exploited on a broad scale to develop next-generation genetic engineering and genome-editing tools. Conveniently, Type II systems are streamlined and rely on a single protein, Cas9, and a guide RNA molecule, comprised of a CRISPR RNA (crRNA) and trans-acting CRISPR RNA (tracrRNA), to achieve effective and programmable nucleic acid targeting and cleavage. Currently, most commercially available Cas9-based genome-editing tools use the CRISPR-Cas system from Streptococcus pyogenes (SpyCas9), although many orthogonal Type II systems are available for diverse and multiplexable genome engineering applications. Here, we discuss the biological significance of Type II CRISPR-Cas elements, including the tracrRNA, crRNA, Cas9, and protospacer-adjacent motif (PAM), and look at the native function of these elements to understand how they can be engineered, enhanced, and optimized for genome editing applications. Additionally, we discuss the basis for orthogonal Cas9 and guide RNA systems that would allow researchers to concurrently use multiple Cas9-based systems for different purposes. Understanding the native function of endogenous Type II CRISPR-Cas systems can lead to new Cas9 tool development to expand the genetic manipulation toolbox.
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Elimination of HIV-1 Genomes from Human T-lymphoid Cells by CRISPR/Cas9 Gene Editing.
We employed an RNA-guided CRISPR/Cas9 DNA editing system to precisely remove the entire HIV-1 genome spanning between 5' and 3' LTRs of integrated HIV-1 proviral DNA copies from latently infected human CD4+ T-cells. Comprehensive assessment of whole-genome sequencing of HIV-1 eradicated cells ruled out any off-target effects by our CRISPR/Cas9 technology that might compromise the integrity of the host genome and further showed no effect on several cell health indices including viability, cell cycle and apoptosis. Persistent co-expression of Cas9 and the specific targeting guide RNAs in HIV-1-eradicated T-cells protected them against new infection by HIV-1. Lentivirus-delivered CRISPR/Cas9 significantly diminished HIV-1 replication in infected primary CD4+ T-cell cultures and drastically reduced viral load in ex vivo culture of CD4+ T-cells obtained from HIV-1 infected patients. Thus, gene editing using CRISPR/Cas9 may provide a new therapeutic path for eliminating HIV-1 DNA from CD4+ T-cells and potentially serve as a novel and effective platform toward curing AIDS.
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Peptide display on small extracellular vesicles directs tissue-specific tropism and delivery of gene editing machinery.
Small extracellular vesicles (EVs) deliver nucleic acid and protein therapeutics that promote tissue repair, however, there are few approaches to direct the tropism of EVs to specific tissues. Here, we address this challenge by engineering EVs to display a peptide library on the surface of EVs that is linked to barcoded guide RNA payloads (gRNAs). We show how these EVs can be administered systemically into mouse models and the cellular uptake of these gRNA-bearing EVs assessed by recovery of the small RNAs followed by PCR amplification and barcode sequencing. Since the design of the peptide library was linked to specific barcodes encoded on the same plasmid, subsequent sequencing of barcode sequences recovered from tissues revealed profiles of EV uptake associated with the display of specific peptide sequences on the surface of EVs. Therefore, candidate peptide motifs were cloned for validation using in vivo and in vitro readouts. In addition to the barcode-based tissue profiling, gRNA-laded EVs mediated functional gene editing in the Cre-loxP R26 LSL-tdTomato reporter mouse model in vivo. These studies demonstrated how EV display linked with barcoded gRNA payloads can address barriers to EV-based delivery of gene editing therapies.
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Generation of homozygous and heterozygous REEP1 knockout induced pluripotent stem cell lines by CRISPR/Cas9 gene editing.
REEP1 is a transmembrane protein in the endoplasmic reticulum (ER) membrane that is involved in shaping and remodeling of the ER. Mutations in REEP1 cause SPG31, an autosomal dominant form of hereditary spastic paraplegia (HSP). Here we show the generation of a homozygous and a heterozygous REEP1 knockout induced pluripotent stem cell line suitable for in vitro disease modelling using the CRISPR/Cas9 editing system.
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Biomedical applications of gene editing.
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High-Throughput Screening to Identify Novel Compounds Affecting the Genome Editing Efficiency of CRISPR System.
Genome editing is a promising therapeutic strategy for genetic disorders by modifying the genome precisely, especially the CRISPR/Cas9 system. However, a major limitation of CRISPR/Cas9 in gene therapy is the biosafety issues caused by off-target effects. Compounds that can modulate the genome editing efficiency of the CRISPR/Cas9 system, especially those reducing the off-target effects, are potentially useful pharmacological tools for improving the effectiveness and safety of genome editing. Here, we performed high-throughput screening in HEK 293FT cells to discover compounds that decrease or increase the genome editing efficiency of the CRISPR/Cas9 system from 9930 compounds. After two rounds of screening, we identified that CP-724714, a ErbB2 (HER2) tyrosine kinase inhibitor, decreased the CRISPR/Cas9 efficiency and reduced the off-target effects by suppressing the efficiency of CRISPR/Cas9, and was thus named a CRISPR decelerator (or inhibitor), while Clofarabine, a DNA synthesis inhibitor, increased the efficiency of CRISPR/Cas9, and was named a CRISPR accelerator. We further identified four compounds (Tranilast, Cerulenin, Rosolic acid and Resveratrol) that affected the efficiency of single-strand annealing (SSA) repair. Among them, Tranilast, Cerulenin and Rosolic acid are potential SSA decelerators, while Resveratrol is a potential SSA accelerator. These identified compounds may be useful in optimizing mammalian genetic manipulation techniques.
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CRISPR FokI Dead Cas9 System: Principles and Applications in Genome Engineering.
The identification of the robust clustered regularly interspersed short palindromic repeats (CRISPR) associated endonuclease (Cas9) system gene-editing tool has opened up a wide range of potential therapeutic applications that were restricted by more complex tools, including zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Nevertheless, the high frequency of CRISPR system off-target activity still limits its applications, and, thus, advanced strategies for highly specific CRISPR/Cas9-mediated genome editing are continuously under development including CRISPR-FokI dead Cas9 (fdCas9). fdCas9 system is derived from linking a FokI endonuclease catalytic domain to an inactive Cas9 protein and requires a pair of guide sgRNAs that bind to the sense and antisense strands of the DNA in a protospacer adjacent motif (PAM)-out orientation, with a defined spacer sequence range around the target site. The dimerization of FokI domains generates DNA double-strand breaks, which activates the DNA repair machinery and results in genomic edit. So far, all the engineered fdCas9 variants have shown promising gene-editing activities in human cells when compared to other platforms. Herein, we review the advantages of all published variants of fdCas9 and their current applications in genome engineering.
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CRISPR/Cas9-mediated gene editing in an exogenous transgene and an endogenous sex determination gene in the Caribbean fruit fly, Anastrepha suspensa.
CRISPR/Cas9-mediated gene-editing, using injected Cas9 protein, was achieved in the Caribbean fruit fly, Anastrepha suspensa, by initially targeting an exogenous transgene, polyubiquitin-regulated EGFP (PUb-EGFP), for heritable non-homologous end-joining (NHEJ) knock-outs using an individual sgRNA. Multiple deletion mutations, ranging from two to five nts proximal to the target site, were identified phenotypically by the loss of green fluorescence in transgenic flies that were also marked with PUb-DsRed. This represented a relatively high efficiency rate of 29% for germ-line mutations. Similar conditions were then used to target an endogenous sex-determination gene, As-transformer-2 (Astra-2), using two sgRNAs that targeted independent exon sequences 671 bp apart. Somatic mutations were identified phenotypically in G0 adult flies at a frequency of 81% based upon intersexual genital morphology, expected to occur only in XX females since Astra-2 knock-outs by dsRNA do not have a phenotypic effect in XY males. Consistent with this expectation, twelve types of short indels, ranging from -15 nts to +5 nts, were identified proximal to the 5' sgRNA-1 target site in intersexual adults. However, the 3' sgRNA-2 target was only associated with a single 774 bp deletion extending from the sgRNA-1 target site to 100 bp downstream of the sgRNA-2 target. This is encouraging for the eventual use of dual target sites for homology-directed repair (HDR) insertions, but suggests that the sgRNA-2 target site tested may not be optimal for Astra-2 HDR modification.
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Applications and explorations of CRISPR/Cas9 in CAR T-cell therapy.
Chimeric antigen receptor(CAR) T-cell therapy has shown remarkable effects and promising prospects in patients with refractory or relapsed malignancies, pending further progress in the next-generation CAR T cells with more optimized structure, enhanced efficacy and reduced toxicities. The clustered regulatory interspaced short palindromic repeat/CRISPR-associated protein 9 (CRISPR/Cas9) technology holds immense promise for advancing the field owing to its flexibility, simplicity, high efficiency and multiplexing in precise genome editing. Herein, we review the applications and explorations of CRISPR/Cas9 technology in constructing allogenic universal CAR T cells, disrupting inhibitory signaling to enhance potency and exploration of safer and more controllable novel CAR T cells.
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