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Light-Driven Activation of RNA-Guided Nucleic Acid Cleavage.
As one of the most favorable stimuli, photoactivation provides an advantageous way to manipulate biological objects. In the current study, we have successfully demonstrated the use of light activation guide RNA (gRNA) strategy for controlling CRISPR systems. By conjugating photolabile protecting groups, the CRISPR functions became minimal, but exposure of acylated gRNAs to 365 nm light triggers the removal of masking groups, leading to the rescue of CRISPR functions. Furthermore, our strategy has been successfully used to control gene editing in human cells. This proof-of-concept study therefore demonstrates the promising potential of our strategy to versatile applications in chemical biology.
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Characterization and applications of Type I CRISPR-Cas systems.
CRISPR-Cas constitutes the adaptive immune system of bacteria and archaea. This RNA-mediated sequence-specific recognition and targeting machinery has been used broadly for diverse applications in a wide range of organisms across the tree of life. The compact class 2 systems, that hinge on a single Cas effector nuclease have been harnessed for genome editing, transcriptional regulation, detection, imaging and other applications, in different research areas. However, most of the CRISPR-Cas systems belong to class 1, and the molecular machinery of the most widespread and diverse Type I systems afford tremendous opportunities for a broad range of applications. These highly abundant systems rely on a multi-protein effector complex, the CRISPR associated complex for antiviral defense (Cascade), which drives DNA targeting and cleavage. The complexity of these systems has somewhat hindered their widespread usage, but the pool of thousands of diverse Type I CRISPR-Cas systems opens new avenues for CRISPR-based applications in bacteria, archaea and eukaryotes. Here, we describe the features and mechanism of action of Type I CRISPR-Cas systems, illustrate how endogenous systems can be reprogrammed to target the host genome and perform genome editing and transcriptional regulation by co-delivering a minimal CRISPR array together with a repair template. Moreover, we discuss how these systems can also be used in eukaryotes. This review provides a framework for expanding the CRISPR toolbox, and repurposing the most abundant CRISPR-Cas systems for a wide range of applications.
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CrispRVariants charts the mutation spectrum of genome engineering experiments.
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Somatic Liver Knockout (SLiK): A Quick and Efficient Way to Generate Liver-Specific Knockout Mice Using Multiplex CRISPR/Cas9 Gene Editing.
Somatic liver knockout (SLiK) is a method developed to rapidly generate a liver-specific knockout of one or several genes. This technique combines the strengths of CRISPR/Cas9 gene editing and hydrodynamic tail-vein injection, a simple in vivo method for transfection of hepatocytes, to harness the powerful selection pressure of tyrosinemic livers to replace host hepatocytes with any desired gene deletion. In this protocol, we will describe sgRNA design and cloning, hydrodynamic tail-vein injection of targeting constructs, and screening and validation methods for efficient in vivo gene editing. © 2020 by John Wiley & Sons, Inc. Support Protocol 1: sgRNA design Support Protocol 2: sgRNA construction: daisy chaining multiple sgRNAs Basic Protocol: Delivery of DNA by hydrodynamic tail-vein injection and liver repopulation of edited hepatocytes Support Protocol 3: Validation of CRISPR/Cas9 cutting in vivo.
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Modeling Breast Cancer Using CRISPR-Cas9-Mediated Engineering of Human Breast Organoids.
Breast cancer is characterized by histological and functional heterogeneity, posing a clinical challenge for patient treatment. Emerging evidence suggests that the distinct subtypes reflect the repertoire of genetic alterations and the target cell. However, the precise initiating events that predispose normal epithelium to neoplasia are poorly understood. Here, we demonstrate that breast epithelial organoids can be generated from human reduction mammoplasties (12 out of 12 donors), thus creating a tool to study the clonal evolution of breast cancer. To recapitulate de novo oncogenesis, we exploited clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 for targeted knockout of four breast cancer-associated tumor suppressor genes (P53, PTEN, RB1, NF1) in mammary progenitor cells from six donors. Mutant organoids gained long-term culturing capacity and formed estrogen-receptor positive luminal tumors on transplantation into mice for one out of six P53/PTEN/RB1-mutated and three out of six P53/PTEN/RB1/NF1-mutated lines. These organoids responded to endocrine therapy or chemotherapy, supporting the potential utility of this model to enhance our understanding of the molecular events that culminate in specific subtypes of breast cancer.
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CRISPR/Cas precision: do we need to worry about off-targeting in plants?
The CRISPR/Cas technology has recently become the tool of choice for targeted genome modification in plants and beyond. Although CRSIPR/Cas offers a rapid and facile way of introducing changes at genomic loci of interest, its application is associated with off-targeting, i.e. introduction of unintended mutations at off-target sites within the genome, which has been reported frequently in the mammalian field. Here we summarise the current knowledge on the precision of CRISPR/Cas in plant systems and provide a summary of state-of the-art strategies for avoiding off-target mutations, as well as unintended on-target changes, in plants. These include using natural (e.g. Cas12a) or engineered (e.g. SpCas9-HF) CRISPR/Cas nucleases characterised by higher precision, as compared to the commonly used wild type SpCas9. In addition, we discuss the usage of CRISPR/Cas nucleases in the form of ribonucleoproteins (RNPs) as an option for reducing off-targeting in plants. Finally, we conclude that the most important factor for reducing CRISPR/Cas off-targeting remains careful selection of target sequences, for which we provide an overview of available online software tools and experimental guidance.
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In vivo CRISPR gene editing in patients with herpetic stromal keratitis.
In vivo CRISPR gene therapy holds large clinical potential, but the safety and efficacy remain largely unknown. Here, we injected a single dose of herpes simplex virus 1 (HSV-1)-targeting CRISPR formulation in the cornea of three patients with severe refractory herpetic stromal keratitis (HSK) during corneal transplantation. Our study is an investigator-initiated, open-label, single-arm, non-randomized interventional trial at a single center (NCT04560790). We found neither detectable CRISPR-induced off-target cleavages by GUIDE-seq nor systemic adverse events for 18 months on average in all three patients. The HSV-1 remained undetectable during the study. Our preliminary clinical results suggest that in vivo gene editing targeting the HSV-1 genome holds acceptable safety as a potential therapy for HSK.
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To CRISPR and beyond: the evolution of genome editing in stem cells.
The goal of editing the genomes of stem cells to generate model organisms and cell lines for genetic and biological studies has been pursued for decades. There is also exciting potential for future clinical impact in humans. While recent, rapid advances in targeted nuclease technologies have led to unprecedented accessibility and ease of gene editing, biology has benefited from past directed gene modification via homologous recombination, gene traps and other transgenic methodologies. Here we review the history of genome editing in stem cells (including via zinc finger nucleases, transcription activator-like effector nucleases and CRISPR-Cas9), discuss recent developments leading to the implementation of stem cell gene therapies in clinical trials and consider the prospects for future advances in this rapidly evolving field.
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Efficient gene targeting in Nicotiana tabacum using CRISPR/SaCas9 and temperature tolerant LbCas12a.
Nicotiana tabacum is a non-food herb that has the potential to be utilized as bio-factory for generating medicines, vaccines or valuable small metabolites. To achieve these goals, the improvement of genetic tools for pre-designed genome modifications is indispensable. The development of CRISPR/Cas nucleases allows the induction of site-specific double-strand breaks to enhance homologous recombination-mediated gene targeting (GT). However, the efficiency of GT is still a challenging obstacle for many crops including tobacco. Recently, studies in several plant species indicated that by replacing SpCas9 with other CRISPR/Cas-based nucleases, GT efficiencies might be enhanced considerably. Therefore, we tested SaCas9 as well as a temperature-insensitive version of LbCas12a (ttLbCas12a) for targeting the tobacco SuRB gene. At the same time, we also optimized the protocol for Agrobacterium-mediated tobacco transformation and tissue culture. In this way, we could improve GT efficiencies to up to a third of the inoculated cotyledons when using ttLbCas12a, which outperformed SaCas9 considerably. In addition, we could show that the conversion tract length of the GT reaction can be up to 606 bp long and in the majority of cases, it is longer than 250 bp. We obtained multiple heritable GT events, mostly heterozygous, but also biallelic GT events and some without T-DNA integration. Thus, we were not only able to obtain CRISPR/Cas-based heritable GT events in allotetraploid Nicotiana tabacum for the first time, but our results also indicate that ttLbCas12a might be a superior alternative for gene editing and GT in tobacco as well as in other crops.
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"Doing CRISPR": The novel case of Atlantic salmon, science and industry.
Salmon farming is a key industry in Norway, with firsthand value of more than 60 billion Norwegian crowns in 2017. The salmon industry is a driving force for biotechnological applications in the marine sector. The recent release of the Atlantic salmon reference genome offers new opportunities to solve major aquaculture bottlenecks that currently limit expansion of the industry. One major bottleneck is the genetic impact of escaped farmed salmon on wild populations. To solve this problem, the industry can use sterile salmon in production. As shown byWargelius et al., sterile salmon can be made by preventing the formation of germ cells through genome editing using the CRISPR-Cas9 method. This approach solves problems of genetic introgression and precocious maturation. However, genome editing of animals, especially for human consumption, raises ethical as well as safety and legal questions. These social and ethical aspects can have tremendous impact in analyzing the final result of salmon farming (e.g., consumer acceptability of a fresh or frozen filet or similar salmon product) but also can be examined "upstream" by describing and assessing the research communities that promote and carry out the science that underpins the salmon industry. Who produces the scientific "facts" that govern the Norwegian aquaculture industry? How do these scientific communities work together? What are the societal impacts of this science? This article uses ethnographical observation and interviews to describe the state-of-the-art of CRISPR gene-editing procedures currently employed in the science and industry collaboration in Norway.
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