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CRISPRi as an efficient tool for gene repression in archaea.
In the years following its discovery and characterization, the CRISPR-Cas system has been modified and converted into a multitude of applications for eukaryotes and bacteria, such as genome editing and gene regulation. Since no such method has been available for archaea, we developed a tool for gene repression in the haloarchaeon Haloferax volcanii by repurposing its endogenous type I-B CRISPR-Cas system. Here, we present the two possible approaches for gene repression as well as our workflow to achieve and assess gene knockdown, offer recommendations on protospacer selection and give some examples of genes we have successfully silenced.
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Progress of CRISPR-Cas Based Genome Editing in Photosynthetic Microbes.
The carbon footprint caused by unsustainable development and its environmental and economic impact has become a major concern in the past few decades. Photosynthetic microbes such as microalgae and cyanobacteria are capable of accumulating value-added compounds from carbon dioxide, and have been regarded as environmentally friendly alternatives to reduce the usage of fossil fuels, thereby contributing to reducing the carbon footprint. This light-driven generation of green chemicals and biofuels has triggered the research for metabolic engineering of these photosynthetic microbes. CRISPR-Cas systems are successfully implemented across a wide range of prokaryotic and eukaryotic species for efficient genome editing. However, the inception of this genome editing tool in microalgal and cyanobacterial species took off rather slowly due to various complications. In this review, we elaborate on the established CRISPR-Cas based genome editing in various microalgal and cyanobacterial species. The complications associated with CRISPR-Cas based genome editing in these species are addressed along with possible strategies to overcome these issues. It is anticipated that in the near future this will result in improving and expanding the microalgal and cyanobacterial genome engineering toolbox.
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Gene editing prospects for treating inherited retinal diseases.
Retinal diseases (RD) include inherited retinal dystrophy (IRD), for example, retinitis pigmentosa and Leber's congenital amaurosis, or multifactorial forms, for example, age-related macular degeneration (AMD). IRDs are clinically and genetically heterogeneous in nature. To date, more than 200 genes are known to cause IRDs, which perturb the development, function and survival of rod and cone photoreceptors or retinal pigment epithelial cells. Conversely, AMD, the most common cause of blindness in the developed world, is an acquired disease of the macula characterised by progressive visual impairment. To date, available therapeutic approaches for RD include nutritional supplements, neurotrophic factors, antiangiogenic drugs for wet AMD and gene augmentation/interference strategy for IRDs. However, these therapies do not aim at correcting the genetic defect and result in inefficient and expensive treatments. The genome editing technology based on clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein (Cas) and an RNA that guides the Cas protein to a predetermined region of the genome, represents an attractive strategy to tackle IRDs without available cure. Indeed, CRISPR/Cas system can permanently and precisely replace or remove genetic mutations causative of a disease, representing a molecular tool to cure a genetic disorder. In this review, we will introduce the mechanism of CRISPR/Cas system, presenting an updated panel of Cas variants and delivery systems, then we will focus on applications of CRISPR/Cas genome editing in the retina, and, as emerging treatment options, in patient-derived induced pluripotent stem cells followed by transplantation of retinal progenitor cells into the eye.
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Integrating gene delivery and gene-editing technologies by adenoviral vector transfer of optimized CRISPR-Cas9 components.
Enhancing the intracellular delivery and performance of RNA-guided CRISPR-Cas9 nucleases (RGNs) remains in demand. Here, we show that nuclear translocation of commonly used Streptococcus pyogenes Cas9 (SpCas9) proteins is suboptimal. Hence, we generated eCas9.4NLS by endowing the high-specificity eSpCas9(1.1) nuclease (eCas9.2NLS) with additional nuclear localization signals (NLSs). We demonstrate that eCas9.4NLS coupled to prototypic or optimized guide RNAs achieves efficient targeted DNA cleavage and probe the performance of SpCas9 proteins with different NLS compositions at target sequences embedded in heterochromatin versus euchromatin. Moreover, after adenoviral vector (AdV)-mediated transfer of SpCas9 expression units, unbiased quantitative immunofluorescence microscopy revealed 2.3-fold higher eCas9.4NLS nuclear enrichment levels than those observed for high-specificity eCas9.2NLS. This improved nuclear translocation yielded in turn robust gene editing after nonhomologous end joining repair of targeted double-stranded DNA breaks. In particular, AdV delivery of eCas9.4NLS into muscle progenitor cells resulted in significantly higher editing frequencies at defective DMD alleles causing Duchenne muscular dystrophy (DMD) than those achieved by AdVs encoding the parental, eCas9.2NLS, protein. In conclusion, this work provides a strong rationale for integrating viral vector and optimized gene-editing technologies to bring about enhanced RGN delivery and performance.
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Development of Tobamovirus-Resistant Tomato Plants by CRISPR-Cas9-Mediated Knockout of Susceptibility Genes.
The advent of genome editing technologies such as CRISPR-Cas9 has revolutionized the development of disease-resistant crops, offering precision and efficiency in targeting specific genetic loci responsible for susceptibility. In this protocol, we harness the CRISPR-Cas9 system to disrupt key susceptibility genes in tomato, aiming to fortify resistance against tobamoviruses, particularly the aggressive tomato brown rugose fruit virus (ToBRFV). By systematically knocking out four TOM1 homologs, genes essential for tobamoviral replication, tomato lines with robust and heritable resistance can be developed while minimizing adverse developmental effects. The approach not only underscores the significance of basic research on host-pathogen interactions in modern crop protection but also lays the groundwork for sustainable, gene-driven resistance strategies in commercial tomato breeding.
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Better living through chemistry: CRISPR/Cas engineered T cells for cancer immunotherapy.
T cells engineered to express transgenes such as chimeric antigen receptors (CAR) or modified T cell receptors (TCR) represent a new pillar of cancer therapy. Use of CRISPR/Cas gene-editing tools now allows even stronger and more precise control over the fate and function of engineered T cell therapies, including multiplex genome editing to facilitate use of off-the-shelf allogeneic T cells and novel approaches which have the potential to overcome some of the limitations of canonical Cas9-mediated DNA cleavage. This review summarizes the CRISPR/Cas techniques that have been used in preclinical research and outlines those that currently being tested in clinical trials.
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Unmarked Gene Editing in Clavibacter michiganensis Using CRISPR/Cas9 and 5-Fluorocytosine Counterselection.
Plant-pathogenic bacteria in the genus Clavibacter are important quarantine species that cause considerable economic loss worldwide. The development of effective gene editing techniques and additional selectable markers is essential to expedite gene functional analysis in this important Gram-positive genus. The current study details a highly efficient unmarked CRISPR/Cas9-mediated gene editing system in Clavibacter michiganensis, which couples the expression of cas9 and single-guide RNA with homology-directed repair templates and the negative selectable marker codA::upp within a single plasmid. Initial experiments indicated that CRISPR/Cas9-mediated transformation could be utilized for both site-directed mutagenesis, in which an A to G point mutation was introduced at the 128th nucleotide of the C. michiganensis rpsL gene to generate a streptomycin-resistant mutant, and complete gene knockout, in which the deletion of the C. michiganensis celA or katA genes resulted in transformants that lacked cellulase and catalase activity, respectively. In subsequent experiments, the introduction of the codA::upp cassette into the transformation vector facilitated the counterselection of unmarked transformants by incubation in the absence of the selective antibiotic, followed by plating on M9 agar containing 5-fluorocytosine at 100 μg/ml, in which an unmarked katA mutant lacking the transformation vector was recovered. Compared with conventional homologous recombination, the unmarked CRISPR/Cas9-mediated system was more useful and convenient because it allowed the template plasmid to be reused repeatedly to facilitate the editing of multiple genes, which constitutes a major advancement that could revolutionize research into C. michiganensis and other Clavibacter spp.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.
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Editing streptomycete genomes in the CRISPR/Cas9 age.
Covering: up to December 2018 This article aims to highlight advantages, drawbacks and issues that users should consider when implementing the use of CRISPR/Cas9-tools for genome editing in streptomycetes, the most prolific source of antimicrobial natural products to date. Here, we examine four toolkits that have so far been made available for streptomycete in vivo-engineering and one for in vitro-editing, and review how they have been applied over the last three years. Our critical evaluation of these toolkits intends to support potential users in determining what they could achieve, what they should consider and what system they should select/optimise for their application.
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Enhanced production of D-pantothenic acid in Corynebacterium glutamicum using an efficient CRISPR-Cpf1 genome editing method.
BACKGROUND: Corynebacterium glutamicum has industrial track records for producing a variety of valuable products such as amino acids. Although CRISPR-based genome editing technologies have undergone immense developments in recent years, the suicide-plasmid-based approaches are still predominant for C. glutamicum genome manipulation. It is crucial to develop a simple and efficient CRISPR genome editing method for C. glutamicum. RESULTS: In this study, we developed a RecombinAtion Prior to Induced Double-strand-break (RAPID) genome editing technology for C. glutamicum, as Cpf1 cleavage was found to disrupt RecET-mediated homologous recombination (HR) of the donor template into the genome. The RAPID toolbox enabled highly efficient gene deletion and insertion, and notably, a linear DNA template was sufficient for gene deletion. Due to the simplified procedure and iterative operation ability, this methodology could be widely applied in C. glutamicum genetic manipulations. As a proof of concept, a high-yield D-pantothenic acid (vitamin B5)-producing strain was constructed, which, to the best of our knowledge, achieved the highest reported titer of 18.62 g/L from glucose only. CONCLUSIONS: We developed a RecET-assisted CRISPR-Cpf1 genome editing technology for C. glutamicum that harnessed CRISPR-induced DSBs as a counterselection. This method is of great importance to C. glutamicum genome editing in terms of its practical applications, which also guides the development of CRISPR genome editing tools for other microorganisms.
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[Type II CRISPR-Cas System Nucleases: A Pipeline for Prediction and In Vitro Characterization].
The use of CRISPR-Cas bacterial adaptive immunity system components for targeted DNA changes has opened broad prospects for programmable genome editing of higher organisms. The most widely used gene editors are based on the Cas9 effectors of the type II CRISPR-Cas systems. In complex with guide RNAs, Cas9 proteins are able to directionally introduce double-stranded breaks into DNA regions that are complementary to guide RNA sequences. Despite the wide range of characterized Cas9s, the search for new Cas9 variants remains an important task, since the available Cas9 editors have several limitations. This paper presents a workflow for the search for and subsequent characterization of new Cas9 nucleases developed in our laboratory. Detailed protocols describing the bioinformatical search, cloning, and isolation of recombinant Cas9 proteins, testing for the presence of nuclease activity in vitro, and determining the PAM sequence, which is required for recognition of DNA targets, are presented. Potential difficulties that may arise, as well as ways to overcome them, are considered.
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