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ARIH2 Ubiquitinates NLRP3 and Negatively Regulates NLRP3 Inflammasome Activation in Macrophages.
The nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome is a molecular platform that induces caspase-1 activation and subsequent IL-1β maturation, and is implicated in inflammatory diseases; however, little is known about the negative regulation of NLRP3 inflammasome activation. In this article, we identified an E3 ligase, Ariadne homolog 2 (ARIH2), as a posttranslational negative regulator of NLRP3 inflammasome activity in macrophages. ARIH2 interacted with NLRP3 via its NACHT domain (aa 220-575) in the NLRP3 inflammasome complex. In particular, we found that while using mutants of ARIH2 and ubiquitin, the really interesting new gene 2 domain of ARIH2 was required for NLRP3 ubiquitination linked through K48 and K63. Deletion of endogenous ARIH2 using CRISPR/Cas9 genome editing inhibited NLRP3 ubiquitination and promoted NLRP3 inflammasome activation, resulting in apoptosis-associated speck-like protein containing a caspase recruitment domain oligomerization, pro-IL-1β processing, and IL-1β production. Conversely, ARIH2 overexpression promoted NLRP3 ubiquitination and inhibited NLRP3 inflammasome activation. Our findings reveal a novel mechanism of ubiquitination-dependent negative regulation of the NLRP3 inflammasome by ARIH2 and highlight ARIH2 as a potential therapeutic target for inflammatory diseases.
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Identification of c.146G > A mutation in a Fabry patient and its correction by customized Cas9 base editors in vitro.
Fabry disease (FD) is a rare X-linked lysosomal storage disorder caused by mutations in the GLA gene, leading to reduced α-galactosidase (α-Gal A) activity. Current treatments, like enzyme replacement, have limitations affecting efficacy and patient outcomes. CRISPR/Cas9 genome editing tools may offer the potential to develop therapeutic strategy via correcting GLA mutations. In this study, we diagnosed a female FD patient with a missense mutation in exon 1 of the GLA gene (c.146G > A, p.R49H). Bioinformatic predictions and biochemical analyses in GLA-knockout cells revealed that this mutation significantly reduced α-Gal A stability and activity, confirming its pathogenicity. To correct this, we used adenine base editing. The mutation, along with a nearby bystander A, was efficiently edited by the traditional N-terminal adenine base editor. To avoid unwanted bystander editing, we developed a series of domain-inlaid base editors with the aim of narrowing editing window. The most effective variant, with deaminase inserted between the 947th and 948th residues of the RUVC3 domain, was further optimized by modifying linker rigidity. These adjustments shifted the editing window, eliminating bystander editing. Our findings clarify the pathogenic nature of a novel GLA mutation and demonstrate the potential of a customized base editor for therapeutic application in FD.
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Precision gene editing: From proof-of-concept to curative therapies.
Gene therapy is evolving from gene addition to precise genome editing, enabling the direct correction of disease-causing mutations. Breakthrough technologies, such as clustered regularly interspaced short palindromic repeats-CRISPR-associated protein (CRISPR-Cas) nucleases, base editors, prime editors, and CRISPR-associated transposases are reshaping the therapeutic landscape. This review covers the progression of precision editing technologies and their clinical applications, spanning from ex vivo therapies to in vivo treatments targeting vital organs. The rise of personalized medicine, highlighted by therapies, such as carbamoyl phosphate synthetase 1 editing, underscores the shift toward N-of-1 medicine for rare diseases. Clinical trial progress, delivery and accessibility challenges, and the role of AI in optimizing editing tools and predicting outcomes are also discussed. These innovations are transforming genetic medicine, offering the promise of safer, more durable, and personalized cures.
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Precise genome-wide base editing by the CRISPR Nickase system in yeast.
The CRISPR/Cas9 system has been applied to efficient genome editing in many eukaryotic cells. However, the bases that can be edited by this system have been limited to those within the protospacer adjacent motif (PAM) and guide RNA-targeting sequences. In this study, we developed a genome-wide base editing technology, "CRISPR Nickase system" that utilizes a single Cas9 nickase. This system was free from the limitation of editable bases that was observed in the CRISPR/Cas9 system, and was able to precisely edit bases up to 53 bp from the nicking site. In addition, this system showed no off-target editing, in contrast to the CRISPR/Cas9 system. Coupling the CRISPR Nickase system with yeast gap repair cloning enabled the construction of yeast mutants within only five days. The CRISPR Nickase system provides a versatile and powerful technology for rapid, site-specific, and precise base editing in yeast.
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Global quantification of off-target activity by base editors.
Base editors are engineered deaminases combined with CRISPR components. These engineered deaminases are designed to target specific sites within DNA or RNA to make a precise change in the molecule. In therapeutics, they hold promise for correcting mutations associated with genetic diseases. However, a key challenge is minimizing unintended edits at off-target sites, which could lead to harmful mutations. Researchers are actively addressing this concern through a variety of optimization efforts that aim to improve the precision of base editors and minimize off-target activity. Here, we examine the various types of off-target activity, and the methods used to evaluate them. Current methods for finding off-target activity focus on identifying similar sequences in the genome or in the transcriptome, assuming the guide RNA misdirects the editor. The main method presented here, that was originally developed to quantify editing levels mediated by the ADAR enzyme, takes a different approach, investigating the inherent activity of base editors themselves, which might lead to off-target edits beyond sequence similarity. The editing index tool quantifies global off-target editing, eliminates the need to detect individual off-target sites, and allows for assessment of the global load of mutations.
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CRISPR base editors: genome editing without double-stranded breaks.
The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 adaptive immunity system has been harnessed for genome editing applications across eukaryotic species, but major drawbacks, such as the inefficiency of precise base editing and off-target activities, remain. A catalytically inactive Cas9 variant (dead Cas9, dCas9) has been fused to diverse functional domains for targeting genetic and epigenetic modifications, including base editing, to specific DNA sequences. As base editing does not require the generation of double-strand breaks, dCas9 and Cas9 nickase have been used to target deaminase domains to edit specific loci. Adenine and cytidine deaminases convert their respective nucleotides into other DNA bases, thereby offering many possibilities for DNA editing. Such base-editing enzymes hold great promise for applications in basic biology, trait development in crops, and treatment of genetic diseases. Here, we discuss recent advances in precise gene editing using different platforms as well as their potential applications in basic biology and biotechnology.
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Development and application of a fast and efficient CRISPR-based genetic toolkit in Bacillus amyloliquefaciens LB1ba02.
BACKGROUND: Bacillus amyloliquefaciens is generally recognized as food safe (GRAS) microbial host and important enzyme-producing strain in the industry. B.amyloliquefaciens LB1ba02 is a production strain suitable for secreting mesophilic α-amylase in the industry. Nevertheless, due to the low transformation efficiency and restriction-modification system, the development of its CRISPR tool lags far behind other species and strains from the genus Bacillus. This work was undertaken to develop a fast and efficient gene-editing tool in B.amyloliquefaciens LB1ba02. RESULTS: In this study, we fused the nuclease-deficient mutant Cas9n (D10A) of Cas9 with activation-induced cytidine deaminase (AID) and developed a fast and efficient base editing system for the first time in B. amyloliquefaciens LB1ba02. The system was verified by inactivating the pyrF gene coding orotidine 5'-phosphate decarboxylase and the mutant could grow normally on M9 medium supplemented with 5-fluoroorotic acid (5-FOA) and uridine (U). Our base editing system has a 6nt editing window consisting of an all-in-one temperature-sensitive plasmid that facilitates multiple rounds of genome engineering in B. amyloliquefaciens LB1ba02. The total editing efficiency of this method reached 100% and it achieved simultaneous editing of three loci with an efficiency of 53.3%. In addition, based on the base editing CRISPR/Cas9n-AID system, we also developed a single plasmid CRISPR/Cas9n system suitable for rapid gene knockout and integration. The knockout efficiency for a single gene reached 93%. Finally, we generated 4 genes (aprE, nprE, wprA, and bamHIR) mutant strain, LB1ba02△4. The mutant strain secreted 1.25-fold more α-amylase into the medium than the wild-type strain. CONCLUSIONS: The CRISPR/Cas9n-AID and CRISPR/Cas9n systems developed in this work proved to be a fast and efficient genetic manipulation tool in a restriction-modification system and poorly transformable strain.
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Highly Efficient Base Editing in Viral Genome Based on Bacterial Artificial Chromosome Using a Cas9-Cytidine Deaminase Fused Protein.
Viruses evolve rapidly and continuously threaten animal health and economy, posing a great demand for rapid and efficient genome editing technologies to study virulence mechanism and develop effective vaccine. We present a highly efficient viral genome manipulation method using CRISPR-guided cytidine deaminase. We cloned pseudorabies virus genome into bacterial artificial chromosome, and used CRISPR-guided cytidine deaminase to directly convert cytidine (C) to uridine (U) to induce premature stop mutagenesis in viral genes. The editing efficiencies were 100%. Comprehensive bioinformatic analysis revealed that a large number of editable sites exist in pseudorabies virus (PRV) genomes. Notably, in our study viral genome exists as a plasmid in E. coli, suggesting that this method is virus species-independent. This application of base-editing provided an alternative approach to generate mutant virus and might accelerate study on virulence and vaccine development.
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A Base Editing Platform Shows Function of Cancer-Associated Variants.
A resource was developed to introduce and interrogate cancer-associated single-nucleotide variants.
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CRISPR Gene Editing of Hematopoietic Stem and Progenitor Cells.
Genetic editing of hematopoietic stem and progenitor cells can be employed to understand gene-function relationships underlying hematopoietic cell biology, leading to new therapeutic approaches to treat disease. The ability to collect, purify, and manipulate primary cells outside the body permits testing of many different gene editing approaches. RNA-guided nucleases, such as CRISPR, have revolutionized gene editing based simply on Watson-Crick base-pairing, employed to direct activity to specific genomic loci. Given the ease and affordability of synthetic, custom RNA guides, testing of precision edits or large random pools in high-throughput screening studies is now widely available. With the ever-growing number of CRISPR nucleases being discovered or engineered, researchers now have a plethora of options for directed genomic change, including single base edits, nicks or double-stranded DNA cuts with blunt or staggered ends, as well as the ability to target CRISPR to other cellular oligonucleotides such as RNA or mitochondrial DNA. Except for single base editing strategies, precise rewriting of larger segments of the genetic code requires delivery of an additional component, templated DNA oligonucleotide(s) encoding the desired changes flanked by homologous sequences that permit recombination at or near the site of CRISPR activity. Altogether, the ever-growing CRISPR gene editing toolkit is an invaluable resource. This chapter outlines available technologies and the strategies for applying CRISPR-based editing in hematopoietic stem and progenitor cells.
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