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A compact Cascade-Cas3 system for targeted genome engineering.
CRISPR-Cas technologies have enabled programmable gene editing in eukaryotes and prokaryotes. However, the leading Cas9 and Cas12a enzymes are limited in their ability to make large deletions. Here, we used the processive nuclease Cas3, together with a minimal Type I-C Cascade-based system for targeted genome engineering in bacteria. DNA cleavage guided by a single CRISPR RNA generated large deletions (7-424 kilobases) in Pseudomonas aeruginosa with near-100% efficiency, while Cas9 yielded small deletions and point mutations. Cas3 generated bidirectional deletions originating from the programmed site, which was exploited to reduce the P. aeruginosa genome by 837 kb (13.5%). Large deletion boundaries were efficiently specified by a homology-directed repair template during editing with Cascade-Cas3, but not Cas9. A transferable 'all-in-one' vector was functional in Escherichia coli, Pseudomonas syringae and Klebsiella pneumoniae, and endogenous CRISPR-Cas use was enhanced with an 'anti-anti-CRISPR' strategy. P. aeruginosa Type I-C Cascade-Cas3 (PaeCas3c) facilitates rapid strain manipulation with applications in synthetic biology, genome minimization and the removal of large genomic regions.
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Delivery technologies for genome editing.
With the recent development of CRISPR technology, it is becoming increasingly easy to engineer the genome. Genome-editing systems based on CRISPR, as well as transcription activator-like effector nucleases (TALENs) and zinc-finger nucleases (ZFNs), are becoming valuable tools for biomedical research, drug discovery and development, and even gene therapy. However, for each of these systems to effectively enter cells of interest and perform their function, efficient and safe delivery technologies are needed. This Review discusses the principles of biomacromolecule delivery and gene editing, examines recent advances and challenges in non-viral and viral delivery methods, and highlights the status of related clinical trials.
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FokI-dCas9 mediates high-fidelity genome editing in pigs.
Gene editing using clustered regularly interspaced short palindromic repeats/Cas9 has great potential for improving the compatibility of porcine organs with human recipients. However, the risk of detrimental off-target mutations in gene-edited pigs remains largely undefined. We have previously generated GGTA1 knock-in pigs for xenotransplantation using FokI-dCas9, a variant of Cas9 that is reported to reduce the frequency of off-target mutagenesis. In this study, we used whole genome sequencing (WGS) and optimized bioinformatic analysis to assess the fidelity of FokI-dCas9 editing in the generation of these pigs. Genomic DNA was isolated from porcine cells before and after gene editing and sequenced by WGS. The genomic sequences were analyzed using GRIDSS variant-calling software to detect putative structural variations (SVs), which were validated by PCR of DNA from knock-in and wild-type pigs. Platypus variant-calling software was used to detect single-nucleotide variations (SNVs) and small insertions/deletions (indels). GRIDSS analysis confirmed the precise integration of one copy of the knock-in construct in the gene-edited cells. Three additional SVs were detected by GRIDSS: deletions in intergenic regions in chromosome 6 and the X chromosome and a duplication of part of the CALD1 gene on chromosome 18. These mutations were not associated with plausible off-target sites, and were not detected in a second line of knock-in pigs generated using the same pair of guide RNAs, suggesting that they were the result of background mutation rather than off-target activity. Platypus identified 1375 SNVs/indels after quality filtering, but none of these were located in proximity to potential off-target sites, indicating that they were probably also spontaneous mutations. This is the first WGS analysis of pigs generated from FokI-dCas9-edited cells. Our results demonstrate that FokI-dCas9 is capable of high-fidelity gene editing with negligible off-target or undesired on-target mutagenesis.
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Seamless knockins in Drosophila via CRISPR-triggered single-strand annealing.
CRISPR-Cas greatly facilitated the integration of exogenous sequences into specific loci. However, knockin generation in multicellular animals remains challenging, partially due to the complexity of insertion screening. Here, we describe SEED/Harvest, a method to generate knockins in Drosophila, based on CRISPR-Cas and the single-strand annealing (SSA) repair pathway. In SEED (from "scarless editing by element deletion"), a switchable cassette is first integrated into the target locus. In a subsequent CRISPR-triggered repair event, resolved by SSA, the cassette is seamlessly removed. Germline excision of SEED cassettes allows for fast and robust knockin generation of both fluorescent proteins and short protein tags in tandem. Tissue-specific expression of Cas9 results in somatic cassette excision, conferring spatiotemporal control of protein labeling and the conditional rescue of mutants. Finally, to achieve conditional protein labeling and manipulation of short tag knockins, we developed a genetic toolbox by functionalizing the ALFA nanobody.
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'Old' genome editors might treat mitochondrial diseases.
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CRISPR/Cas9-edited NSG mice as PDX models of human leukemia to address the role of niche-derived SPARC.
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An overview on in-vivo generation of CAR-T cells using CRISPR-loaded functionalized nanocarriers for treating B-cell lineage acute lymphoblastic leukemia.
Chimeric antigen receptor T (CAR-T) cell therapy has become a milestone in the management of B cell lineage acute lymphoblastic leukemia. Yet, the traditional method-dependent on ex vivo manipulation, amplification, and reinfusion of autologous T cells-is high-cost, low-scalability, and severely immune-related toxicity. Here, we report a new nano-immunoengineering platform that allows in vivo production of chimeric antigen receptor T cells through the use of functionalized nanoparticles carrying clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) gene editing elements. These nanoparticles are engineered to specifically target blood circulating T lymphocytes and deliver CRISPR/Cas9 complexes that have the ability to integrate chimeric antigen receptor constructs into the TRAC locus and knock out immune checkpoint genes like programmed cell death protein 1 (PD-1) simultaneously. Targeted delivery, endosomal escape, and efficient genome editing with minimal off-target effects are ensured through gold-based and DNA nanostructure-based carriers. Preclinical models show effective in vivo programming of functional chimeric antigen receptor T cells with vigorous antitumor efficacy, improved persistence, and decreased cytokine release syndrome. This method is a revolutionary breakthrough in cancer immunotherapy that provides a scalable, economical, and clinically flexible replacement for conventional chimeric antigen receptor T cell production.
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What have we learned recently from transgenic mouse models about neurodegeneration? The most promising discoveries of this millennium.
Neurodegenerative diseases are currently a major challenge in elderly care due to demographic changes and dramatic increases in the aging population worldwide. This review is focused on the most promising research discovering ways to attenuate neural loss or enhance neuroprotection and unraveling the basis of neurodegeneration using transgenic mouse models. With the recent introduction of the powerful and relatively simple gene-editing tool CRISPR-Cas9, we have entered a new era in genetic engineering that will certainly lead to a variety of new transgenic models in the near future. The aim of this review is to note the most interesting avenues addressing unmet needs in neurodegenerative disease research that could provide promising targets for both the development of new models and the study of existing ones.
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CRISPR-PLANT v2: an online resource for highly specific guide RNA spacers based on improved off-target analysis.
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Bioproduction process of natural products and biopharmaceuticals: Biotechnological aspects.
Decades of research have been put in place for developing sustainable routes of bioproduction of high commercial value natural products (NPs) on the global market. In the last few years alone, we have witnessed significant advances in the biotechnological production of NPs. The development of new methodologies has resulted in a better understanding of the metabolic flux within the organisms, which have driven manipulations to improve production of the target product. This was further realised due to the recent advances in the omics technologies such as genomics, transcriptomics, proteomics, metabolomics and secretomics, as well as systems and synthetic biology. Additionally, the combined application of novel engineering strategies has made possible avenues for enhancing the yield of these products in an efficient and economical way. Invention of high-throughput technologies such as next generation sequencing (NGS) and toolkits for genome editing Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated 9 (CRISPR/Cas9) have been the game changers and provided unprecedented opportunities to generate rationally designed synthetic circuits which can produce complex molecules. This review covers recent advances in the engineering of various hosts for the production of bioactive NPs and biopharmaceuticals. It also highlights general approaches and strategies to improve their biosynthesis with higher yields in a perspective of plants and microbes (bacteria, yeast and filamentous fungi). Although there are numerous reviews covering this topic on a selected species at a time, our approach herein is to give a comprehensive understanding about state-of-art technologies in different platforms of organisms.
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