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CRISPRedict: a CRISPR-Cas9 web tool for interpretable efficiency predictions.
The development of the CRISPR-Cas9 technology has provided a simple yet powerful system for genome editing. Current gRNA design tools serve as an important platform for the efficient application of the CRISPR systems. However, most of the existing tools are black-box models that suffer from limitations, such as variable performance and unclear mechanism of decision making. Here, we introduce CRISPRedict, an interpretable gRNA efficiency prediction model for CRISPR-Cas9 gene editing. Its strength lies in the fact that it can accurately predict efficient guide RNAs-with equivalent performance to state-of-the-art tools-while being a simple linear model. Implemented as a user-friendly web server, CRISPRedict offers (i) quick and accurate predictions across various experimental conditions (e.g. U6/T7 transcription); (ii) regression and classification models for scoring gRNAs and (iii) multiple visualizations to explain the obtained results. Given its performance, interpretability, and versatility, we expect that it will assist researchers in the gRNA design process and facilitate genome editing research. CRISPRedict is available for use at http://www.crispredict.org/.
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Production of a Chimeric Mouse-Fish Monoclonal Antibody by the CRISPR/Cas9 Technology.
The CRISPR/Cas9 system, a defense mechanism naturally occurring in prokaryotes, has been recently repurposed as an RNA-guided DNA targeting platform and widely used as a powerful tool for genome editing. Here we describe how to modify the carboxy-terminal region, called Fragment crystallizable (Fc) region, of a murine monoclonal antibody by replacing the heavy chain constant exons with those from a teleost fish antibody by the CRISPR/Cas9 system. We outline optimal conditions for knockout and knockin mechanisms to edit the Immunoglobulin heavy chain (IgH) constant region gene locus in a murine hybridoma cell line. A chimeric mouse-fish monoclonal antibody can be successfully produced by hybridoma cell lines engineered according to this protocol.
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Emerging applications of CRISPR/Cas9 gene editing technology in reversing drug resistance in oral squamous cell carcinoma.
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On- and off-target effects of paired CRISPR-Cas nickase in primary human cells.
Undesired on- and off-target effects of CRISPR-Cas nucleases remain a challenge in genome editing. While the use of Cas9 nickases has been shown to minimize off-target mutagenesis, their use in therapeutic genome editing has been hampered by a lack of efficacy. To overcome this limitation, we and others have developed double-nickase-based strategies to generate staggered DNA double-strand breaks to mediate gene disruption or gene correction with high efficiency. However, the impact of paired single-strand nicks on genome integrity has remained largely unexplored. Here, we developed a novel CAST-seq pipeline, dual CAST, to characterize chromosomal aberrations induced by paired CRISPR-Cas9 nickases at three different loci in primary keratinocytes derived from patients with epidermolysis bullosa. While targeting COL7A1, COL17A1, or LAMA3 with Cas9 nucleases caused previously undescribed chromosomal rearrangements, no chromosomal translocations were detected following paired-nickase editing. While the double-nicking strategy induced large deletions/inversions within a 10 kb region surrounding the target sites at all three loci, similar to the nucleases, the chromosomal on-target aberrations were qualitatively different and included a high proportion of insertions. Taken together, our data indicate that double-nickase approaches combine efficient editing with greatly reduced off-target effects but still leave substantial chromosomal aberrations at on-target sites.
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Gene targeting in adult organs using in vivo cleavable donor plasmids for CRISPR-Cas9 and CRISPR-Cas12a.
The CRISPR-Cas system for in vivo genome editing is a powerful tool for gene therapy against several diseases. We have previously developed the pCriMGET_9-12a system, an in vivo cleavable donor plasmid for precise targeted knock-in of exogenous DNA by both Cas9 and Cas12a. Here, we show that the pCriMGET_9-12a system can be applied for in vivo in-frame knock-in of exogenous DNA in adult mouse liver by hydrodynamic delivery of the targeting plasmids. The in vivo cleavable pCriMGET_9-12a donor plasmids significantly increased the knock-in efficiency of both CRISPR-Cas9 and CRISPR-Cas12a in the adult mouse liver compared to uncleavable donor plasmids. This strategy also achieved in-frame reporter gene knock-in without indel mutations. Therefore, in vivo gene targeting using the pCriMGET_9-12a system may contribute to the establishment of safer, more precise, versatile and efficient gene therapy methods in adult organs.
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Lipopolymer Nanoparticle-Mediated In Vivo CRISPR-Cas9 Editing of Mrg15 Restores Mitochondrial Mitophagy to Alleviate Metabolic-Associated Steatohepatitis.
Metabolic-associated steatohepatitis (MASH) remains difficult to treat due to the lack of interventions capable of targeting upstream disease drivers and achieving durable disease modification. The epigenetic regulator Mrg15 has been implicated in mitochondrial dysfunction and metabolic stress in the liver, suggesting its potential relevance to MASH pathogenesis. Here, we develop a liver-targeted lipopolymer nanoparticle (LPNP)-mediated gene editing platform to enable in vivo disruption of Mrg15 by co-delivery of Cas9 mRNA and Mrg15 sgRNA. The screened P64H/Mrg15 system achieved efficient hepatic delivery and genome editing, resulting in reduced Mrg15 expression in hepatocytes. In the MASH mouse model, P64H/Mrg15 treatment was associated with decreased hepatic lipid accumulation, improved liver injury markers, and attenuation of inflammation and fibrosis. Sequence-level analyses confirmed on-target editing in liver tissue, and systemic histopathological evaluation revealed no overt toxicity in major organs under the tested dosing regimen. Transcriptomic profiling revealed coordinated pathway-level associations involving metabolic, inflammatory, and autophagy-related regulation, while protein-level analyses demonstrated alterations in selected autophagy- and mitophagy-related regulators, including TUFM and LC3B-II. Together, these findings identify Mrg15 as a disease-relevant epigenetic regulator in MASH and highlight liver-directed P64H LPNP/CRISPR delivery as a promising non-viral strategy for modulating upstream regulatory pathways in metabolic liver disease.
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[Application and optimization of CRISPR/Cas system in bacteria].
Clustered regular interspaced short palindromic repeats (CRISPR) system has been widely used in recent years. Compared with traditional genome editing technology, CRISPR/Cas system has notable advantages, including high editing efficiency, high specificity, low cost and the convenience for manipulation. Type Ⅱ and Ⅴ CRISPR/Cas system only requires a single Cas9 protein or a single Cpf1 protein as effector nucleases for cutting double-stranded DNA, developed as genome editing tools. At present, CRISPR/Cas9 technology has been successfully applied to the genome editing of eukaryotes such as zebrafish, mice and human cells, whereas limited progress has been made in the genome editing of bacteria. In our review, we describe CRISPR/Cas system, its mechanism and summarize the optimization and progress of genome editing in bacteria.
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Gene Editing, Enhancing and Women's Role.
A recent article on the front page of The Independent (September 18, 2015) reported that the genetic 'manipulation' of IVF embryos is to start in Britain, using a new revolutionary gene-editing technique, called Crispr/Cas9. About three weeks later (Saturday 10, October 2015), on the front page of the same newspaper, it was reported that the National Health Service (NHS) faces a one billion pound deficit only 3 months into the new year. The hidden connection between these reports is that gene editing could be used to solve issues related to health care allocation. Improving the health of future generations might coincide with public health goals; it might improve the health of individuals and communities, and, if successful, might be seen as a public good. However, enhancing future generations will require In Vitro Fertilisation and Pre-implantation Genetic Diagnosis. Remarkably, the necessary involvement of women in an enhancing scenario has not been discussed by its proponents. The present discourse on moral obligations of future generations, although not referring to women, seems to imply that women might be required, morally, if not legally, to reproduce with IVF. Enhancing future generations will be gendered, unless the artificial womb is developed. These are challenging issues that require a wider perspective, of both women and men. Despite the lack of a unified feminist conclusion in the discussions about the merits and risks of human genome modification, there is an urgent need to clarify the role of women in this scenario.
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Autonomous Feedback-Driven Engineered DNAzyme-Coated Trojan Horse-like Nanocapsules for On-Demand CRISPR/Cas9 Delivery.
Manipulating the expression of cellular genes through efficient CRISPR/Cas9 delivery is rapidly evolving into a desirable tumor therapeutics. The exposure of CRISPR/Cas9 to a complex external environment poses challenges for conventional delivery carriers in achieving responsive and accurate release. Here, we report a Trojan horse-like nanocapsule for the on-demand delivery of CRISPR/Cas9 in a microRNA-responsive manner, enabling precise tumor therapy. The nanocapsule comprises a nanoassembled, engineered DNAzyme shell encasing a Cas9/sgRNA complex core. The DNAzyme, functioning as a catalytic unit, undergoes a conformational change in the presence of tumor-associated microRNA, followed by activating a positive feedback-driven autonomous catabolic cycle of the nanocapsule shell. This catabolic cycle is accomplished through chain reactions of DNAzyme "cleavage-hybridization-cleavage", which ensures sensitivity in microRNA recognition and effective release of Cas9/sgRNA. Utilizing this Trojan horse-like nanocapsule, as low as 1.7 pM microRNA-21 can trigger the on-demand release of Cas9/sgRNA, enabling the specific editing of the protumorigenic microRNA coding gene. The resulting upregulation of tumor suppressor genes induces apoptosis in tumor cells, leading to significant inhibition of tumor growth by up to 75.94%. The Trojan horse-like nanocapsule, with superior programmability and biocompatibility, is anticipated to serve as a promising carrier for tailoring responsive gene editing systems, achieving enhanced antitumor specificity and efficacy.
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Gene editing in the context of an increasingly complex genome.
The reporting of the first draft of the human genome in 2000 brought with it much hope for the future in what was felt as a paradigm shift toward improved health outcomes. Indeed, we have now mapped the majority of variation across human populations with landmark projects such as 1000 Genomes; in cancer, we have catalogued mutations across the primary carcinomas; whilst, for other diseases, we have identified the genetic variants with strongest association. Despite this, we are still awaiting the genetic revolution in healthcare to materialise and translate itself into the health benefits for which we had hoped. A major problem we face relates to our underestimation of the complexity of the genome, and that of biological mechanisms, generally. Fixation on DNA sequence alone and a 'rigid' mode of thinking about the genome has meant that the folding and structure of the DNA molecule -and how these relate to regulation- have been underappreciated. Projects like ENCODE have additionally taught us that regulation at the level of RNA is just as important as that at the spatiotemporal level of chromatin.In this review, we chart the course of the major advances in the biomedical sciences in the era pre- and post the release of the first draft sequence of the human genome, taking a focus on technology and how its development has influenced these. We additionally focus on gene editing via CRISPR/Cas9 as a key technique, in particular its use in the context of complex biological mechanisms. Our aim is to shift the mode of thinking about the genome to that which encompasses a greater appreciation of the folding of the DNA molecule, DNA- RNA/protein interactions, and how these regulate expression and elaborate disease mechanisms.Through the composition of our work, we recognise that technological improvement is conducive to a greater understanding of biological processes and life within the cell. We believe we now have the technology at our disposal that permits a better understanding of disease mechanisms, achievable through integrative data analyses. Finally, only with greater understanding of disease mechanisms can techniques such as gene editing be faithfully conducted.
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