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Highly specific epigenome editing by CRISPR-Cas9 repressors for silencing of distal regulatory elements.
Epigenome editing with the CRISPR (clustered, regularly interspaced, short palindromic repeats)-Cas9 platform is a promising technology for modulating gene expression to direct cell phenotype and to dissect the causal epigenetic mechanisms of gene regulation. Fusions of nuclease-inactive dCas9 to the Krüppel-associated box (KRAB) repressor (dCas9-KRAB) can silence target gene expression, but the genome-wide specificity and the extent of heterochromatin formation catalyzed by dCas9-KRAB are not known. We targeted dCas9-KRAB to the HS2 enhancer, a distal regulatory element that orchestrates the expression of multiple globin genes, and observed highly specific induction of H3K9 trimethylation (H3K9me3) at the enhancer and decreased chromatin accessibility of both the enhancer and its promoter targets. Targeted epigenetic modification of HS2 silenced the expression of multiple globin genes, with minimal off-target changes in global gene expression. These results demonstrate that repression mediated by dCas9-KRAB is sufficiently specific to disrupt the activity of individual enhancers via local modification of the epigenome.
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[Advances in utilizing the endogenous CRISPR-Cas system for genome editing of lactic acid bacteria].
The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated proteins) system is an efficient and precise gene editing tool. The development of this technology has promoted genome editing into a rapid development stage. The most widely used Cas9 protein is SpyCas9 from Streptococcus pyogenes. As a "gene scissors", the SpyCas9 protein is widely used in eukaryotes including mammals and plants. However, the application of this protein in some lactic acid bacteria (LAB) is still hampered by many factors. It has been identified that LAB genomes possess many types of CRISPR system and are rich in uncharacterized Cas proteins. Genome editing of LAB is possible by repurposing the endogenous CRISPR-Cas systems in LAB combined with exogenous single guide RNA (sgRNA) and homologous recombination template. This method employs its endogenous CRISPR-Cas system for gene editing, which has the advantages including easiness for transformation due to the relatively small targeting vector, and no concern about the toxicity of heterologous Cas9 to host cells. Compared to CRISPR-SpyCas9, the endogenous CRISPR-Cas system is more suitable for genome editing of LAB, and it may become the main genome editing tool for some LAB in the future. This article summarizes the advances in this field.
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Emerging Role of CRISPR/Cas9 Technology for MicroRNAs Editing in Cancer Research.
MicroRNAs (miRNA) are small, noncoding RNA molecules with a master role in the regulation of important tasks in different critical processes of cancer pathogenesis. Because there are different miRNAs implicated in all the stages of cancer, for example, functioning as oncogenes, this makes these small molecules suitable targets for cancer diagnosis and therapy. RNA-mediated interference has been one major approach for sequence-specific regulation of gene expression in eukaryotic organisms. Recently, the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system, first identified in bacteria and archaea as an adaptive immune response to invading genetic material, has been explored as a sequence-specific molecular tool for editing genomic sequences for basic research in life sciences and for therapeutic purposes. There is growing evidence that small noncoding RNAs, including miRNAs, can be targeted by the CRISPR/Cas9 system despite their lacking an open reading frame to evaluate functional loss. Thus, CRISPR/Cas9 technology represents a novel gene-editing strategy with compelling robustness, specificity, and stability for the modification of miRNA expression. Here, I summarize key features of current knowledge of genomic editing by CRISPR/Cas9 technology as a feasible strategy for globally interrogating miRNA gene function and miRNA-based therapeutic intervention. Alternative emerging strategies for nonviral delivery of CRISPR/Cas9 core components into human cells in a clinical context are also analyzed critically. Cancer Res; 77(24); 6812-7. ©2017 AACR.
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Unleashing the Therapeutic Potential of CAR-T Cell Therapy Using Gene-Editing Technologies.
Chimeric antigen receptor (CAR) T-cell therapy, an emerging immunotherapy, has demonstrated promising clinical results in hematological malignancies including B-cell malignancies. However, accessibility to this transformative medicine is highly limited due to the complex process of manufacturing, limited options for target antigens, and insufficient anti-tumor responses against solid tumors. Advances in gene-editing technologies, such as the development of Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9), have provided novel engineering strategies to address these limitations. Development of next-generation CAR-T cells using gene-editing technologies would enhance the therapeutic potential of CAR-T cell treatment for both hematologic and solid tumors. Here we summarize the unmet medical needs of current CAR-T cell therapies and gene-editing strategies to resolve these challenges as well as safety concerns of gene-edited CAR-T therapies.
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Genome-Wide Analysis of Off-Target CRISPR/Cas9 Activity in Single-Cell-Derived Human Hematopoietic Stem and Progenitor Cell Clones.
CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9)-mediated genome editing holds remarkable promise for the treatment of human genetic diseases. However, the possibility of off-target Cas9 activity remains a concern. To address this issue using clinically relevant target cells, we electroporated Cas9 ribonucleoprotein (RNP) complexes (independently targeted to two different genomic loci, the CXCR4 locus on chromosome 2 and the AAVS1 locus on chromosome 19) into human mobilized peripheral blood-derived hematopoietic stem and progenitor cells (HSPCs) and assessed the acquisition of somatic mutations in an unbiased, genome-wide manner via whole genome sequencing (WGS) of single-cell-derived HSPC clones. Bioinformatic analysis identified >20,000 total somatic variants (indels, single nucleotide variants, and structural variants) distributed among Cas9-treated and non-Cas9-treated control HSPC clones. Statistical analysis revealed no significant difference in the number of novel non-targeted indels among the samples. Moreover, data analysis showed no evidence of Cas9-mediated indel formation at 623 predicted off-target sites. The median number of novel single nucleotide variants was slightly elevated in Cas9 RNP-recipient sample groups compared to baseline, but did not reach statistical significance. Structural variants were rare and demonstrated no clear causal connection to Cas9-mediated gene editing procedures. We find that the collective somatic mutational burden observed within Cas9 RNP-edited human HSPC clones is indistinguishable from naturally occurring levels of background genetic heterogeneity.
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Conditional Control of CRISPR/Cas9 Function by Chemically Modified Oligonucleotides.
The CRISPR (clustered regularly interspaced short palindromic repeats) system has emerged as a revolutionary gene-editing tool with immense potential in gene therapy, functional genomics, and beyond. However, achieving precise spatiotemporal control of gene editing in specific cells and tissues while effectively mitigating potential risks, such as off-target effects, remains a key challenge for its clinical translation. To overcome these limitations, researchers have developed innovative strategies based on chemical modifications of oligonucleotides to enhance the precision, efficiency, and controllability of CRISPR/Cas9-mediated gene editing. By introducing conditional responsive elements, such as photosensitive groups, small-molecule responsive units, and supramolecular structures, they have successfully achieved precise spatiotemporal and dose-dependent regulation of CRISPR/Cas9 function. This review provides a comprehensive overview of recent advancements in gRNA regulation strategies based on chemical modifications of oligonucleotides, discussing their applications in improving the efficiency, specificity, and controllability of CRISPR/Cas9 editing. We also highlight the challenges associated with the conditional control of gRNA and offer insights into future directions for the chemical regulation of gRNA to further advance CRISPR/Cas9 technology.
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Cytoprotective role of autophagy against BH3 mimetic gossypol in ATG5 knockout cells generated by CRISPR-Cas9 endonuclease.
Previously, we demonstrated the association between autophagy and gossypol-induced growth inhibition of mutant BRAF melanoma cells. Here, we investigate the role of autophagy in ATG5 knockout cell lines generated by the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas-mediated genome editing. The MTT assay revealed that the inhibitory effect of gossypol was weaker on ATG5 knockout cells than that on the wild type (WT) cells. The conversion of non-autophagic LC3-I to autophagic LC3-II and RT-PCR confirmed the functional gene knockout. However, Cyto-ID autophagy assay revealed that gossypol induced ATG5- and LC3-independent autophagy in ATG5 knockout cells. Moreover, gossypol acts as an autophagy inducer in ATG5 knockout cells while blocking the later stages of the autophagy process in WT cells, which was determined by measuring autophagic flux after co-treatment of gossypol with chloroquine (late-stage autophagy inhibitor). On the other hand, inhibition of autophagy with 3-MA or Beclin-1 siRNA caused a partial increase in the sensitivity to gossypol in ATG5 knockout cells, but not in the WT cells. Together, our findings suggest that the resistance to gossypol in ATG5 knockout cells is associated with increased cytoprotective autophagy, independent of ATG5.
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Revolutionizing cattle breeding: Gene editing advancements for enhancing economic traits.
Beef and dairy products are rich in protein and amino acids, making them highly nutritious for human consumption. The increasing use of gene editing technology in agriculture has paved the way for genetic improvement in cattle breeding via the development of the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) system. Gene sequences are artificially altered and employed in the pursuit of improving bovine breeding research through targeted knockout, knock-in, substitution, and mutation methods. This review offers a comprehensive analysis of the advancements in gene editing technology and its diverse applications in enhancing both quantitative and qualitative traits across livestock. These applications encompass areas such as meat quality, milk quality, fertility, disease resistance, environmental adaptability, sex control, horn development, and coat colour. Furthermore, the review considers prospective ideas and insights that may be employed to refine breeding traits, enhance editing efficiency, and navigate the ethical considerations associated with these advancements. The review's focus on improving the quality of beef and milk is intended to enhance the economic viability of these products. Furthermore, it constitutes a valuable resource for scholars and researchers engaged in the fields of cattle genetic improvement and breeding.
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Overcoming the Delivery Challenges in CRISPR/Cas9 Gene Editing for Effective Cancer Treatment: A Review of Delivery Systems.
Therapeutic strategies based on gene editing provide the ability to modify faulty genes contributing to the development of diseases such as cancer by directly altering the cellular machinery. The clustered regularly interspaced short palindromic repeats associated nuclease 9 (CRISPR/Cas9) system is currently the primary tool used for gene editing. Several effective Cas9 variants have already been established to address the complex genetic modifications that arise during diseases. Although gene-editing systems have made significant advancements, a primary obstacle that requires attention is the transportation of CRISPR/Cas to diverse target cells, both in vivo and in vitro, to render them suitable for clinical implementation. Various strategies can be utilized to facilitate the transportation of the CRISPR/Cas systems into mammalian cells. Herein, we reviewed contemporary research about delivery systems for gene-editing systems that interact effectively in biological systems. This review explores the benefits and drawbacks of using extracellular vesicles and viral vectors as vehicles for delivering the CRISPR/Cas system in the context of cancer treatment.
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CRISPR/Cas9: Principle, Applications, and Delivery through Extracellular Vesicles.
The establishment of CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) technology for eukaryotic gene editing opened up new avenues not only for the analysis of gene function but also for therapeutic interventions. While the original methodology allowed for targeted gene disruption, recent technological advancements yielded a rich assortment of tools to modify genes and gene expression in various ways. Currently, clinical applications of this technology fell short of expectations mainly due to problems with the efficient and safe delivery of CRISPR/Cas9 components to living organisms. The targeted in vivo delivery of therapeutic nucleic acids and proteins remain technically challenging and further limitations emerge, for instance, by unwanted off-target effects, immune reactions, toxicity, or rapid degradation of the transfer vehicles. One approach that might overcome many of these limitations employs extracellular vesicles as intercellular delivery devices. In this review, we first introduce the CRISPR/Cas9 system and its latest advancements, outline major applications, and summarize the current state of the art technology using exosomes or microvesicles for transporting CRISPR/Cas9 constituents into eukaryotic cells.
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