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The Diversifying Distribution Trends of Maturing CRISPR Technologies by Addgene.
Since the advent of Cas9-based CRISPR technologies in 2012, there has been a remarkable growth in genome editing research, literature, applications, and translational impact. Much of this research has been fueled by the global dissemination of CRISPR plasmids through nonprofit distribution by Addgene, as both a repository and distributor of enabling biological material. Recently, key milestones have been reached, with over 20,000 plasmids deposited by over 1,000 labs, being distributed over 300,000 times globally. The driving trends reflect multidimensional diversification in terms of effectors (Cas9, Cas12 and beyond), editing modalities (base editing, prime editing, epigenetic modification, CRISPRi/a), and deployment across phylogenetic groups (mammalian, bacterial, plant, yeast, insects, and more). Noteworthy, guide RNA and HDR templates account for the bulk of deposits, while cloning backbones are the most requested, and lentiviral plasmids comprise the majority of expression material. The data reflect a continued diversification of the CRISPR-based toolbox, robust interest in genome editing applications across the tree of life, maturation in terms of adoption, and rising relative distribution beyond the USA and China, with Addgene continuing to play a critical role in access to equitable and disruptive technologies.
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CRISPR-Cas in its prime.
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Rewired Cas9s with Minimal Sequence Constraints.
The genome editing toolkit is ever expanding. Although CRISPR-Cas systems can target virtually any gene, single-nucleotide resolution is yet to be achieved. Walton and colleagues engineered nucleases and base editors compatible with every protospacer adjacent motif (PAM) to achieve high-precision targeting. Their findings revealed the striking plasticity of Cas9.
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CRISPR inactivation by integration.
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Improving Plant Genome Editing with High-Fidelity xCas9 and Non-canonical PAM-Targeting Cas9-NG.
Two recently engineered SpCas9 variants, namely xCas9 and Cas9-NG, show promising potential in improving targeting specificity and broadening the targeting range. In this study, we evaluated these Cas9 variants in the model and crop plant, rice. We first tested xCas9-3.7, the most effective xCas9 variant in mammalian cells, for targeted mutagenesis at 16 possible NGN PAM (protospacer adjacent motif) combinations in duplicates. xCas9 exhibited nearly equivalent editing efficiency to wild-type Cas9 (Cas9-WT) at most canonical NGG PAM sites tested, whereas it showed limited activity at non-canonical NGH (H = A, C, T) PAM sites. High editing efficiency of xCas9 at NGG PAMs was further demonstrated with C to T base editing by both rAPOBEC1 and PmCDA1 cytidine deaminases. With mismatched sgRNAs, we found that xCas9 had improved targeting specificity over the Cas9-WT. Furthermore, we tested two Cas9-NG variants, Cas9-NGv1 and Cas9-NG, for targeting NGN PAMs. Both Cas9-NG variants showed higher editing efficiency at most non-canonical NG PAM sites tested, and enabled much more efficient editing than xCas9 at AT-rich PAM sites such as GAT, GAA, and CAA. Nevertheless, we found that Cas9-NG variants showed significant reduced activity at the canonical NGG PAM sites. In stable transgenic rice lines, we demonstrated that Cas9-NG had much higher editing efficiency than Cas9-NGv1 and xCas9 at NG PAM sites. To expand the base-editing scope, we developed an efficient C to T base-editing system by making fusion of Cas9-NG nickase (D10A version), PmCDA1, and UGI. Taken together, our work benchmarked xCas9 as a high-fidelity nuclease for targeting canonical NGG PAMs and Cas9-NG as a preferred variant for targeting relaxed PAMs for plant genome editing.
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PrimeRoot for targeted large DNA insertion in plants.
Genome editing technologies such as clustered regularly interspaced short palindromic repeats (CRISPR) have revolutionized plant breeding through targeted genome and transcriptome modifications. However, accurate insertion of large DNA cargoes remains challenging. Recently, Sun and colleagues introduced PrimeRoot, a groundbreaking technology that enables precise and targeted integration of large DNA cargoes into plant genomes with remarkable efficiency and accuracy.
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Role of CRISPR-Cas systems in periodontal disease pathogenesis and potential for periodontal therapy: A review.
Clustered regularly interspaced short palindromic repeats (CRISPRs) are DNA sequences capable of editing a host genome sequence. CRISPR and its specific CRISPR-associated (Cas) protein complexes have been adapted for various applications. These include activating or inhibiting specific genetic sequences or acting as molecular scissors to cut and modify the host DNA precisely. CRISPR-Cas systems are also naturally present in many oral bacteria, where they aid in nutrition, biofilm formation, inter- and intraspecies communication (quorum sensing), horizontal gene transfer, virulence, inflammation modulation, coinfection, and immune response evasion. It even functions as an adaptive immune system, defending microbes against invading viruses and foreign genetic elements from other bacteria by targeting and degrading their DNA. Recently, CRISPR-Cas systems have been tested as molecular editing tools to manipulate specific genes linked with periodontal disease (such as periodontitis) and as novel methods of delivering antimicrobial agents to overcome antimicrobial resistance. With the rapidly increasing role of CRISPR in treating inflammatory diseases, its application in periodontal disease is also becoming popular. Therefore, this review aims to discuss the different types of CRISPR-Cas in oral microbes and their role in periodontal disease pathogenesis and precision periodontal therapy.
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My CRISPR Story: Back to Brazil.
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[Research advance and application in the gene therapy of gene editing technologies].
Gene editing technologies are used to specifically edit the target sequence. With the development of zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), regular clustering of short palindrome repeats (CRISPR) and single base editing (BE) techniques, gene editing technologies not only provide powerful tools for gene functional studies, but also offer new therapeutic strategies in biomedical research. Gene editing has demonstrated broad application prospects in the gene therapy field, as well as in the construction of animal and cell models, drug target screening and gene functional research. In this review, we summarize several typical gene editing technologies, their characteristics and applications in gene therapy and discusses their opportunities and challenges in gene therapy, thereby providing critical insights and references on the clinical application of gene editing technologies.
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CRISPR alleviates muscular dystrophy in dogs.
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