Revolutionizing in vivo therapy with CRISPR/Cas genome editing: breakthroughs, opportunities and challenges
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PROVIDER: S-EPMC10867117 | biostudies-literature | 2024 Jan
REPOSITORIES: biostudies-literature
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Project description:CRISPR/Cas9-based genome editing has quickly emerged as a powerful breakthrough technology for use in diverse settings across biomedical research and therapeutic development. Recent efforts toward understanding gene modification methods in vitro have led to substantial improvements in ex vivo genome editing efficiency. Because disease targets for genomic correction are often localized in specific organs, realization of the full potential of genomic medicines will require delivery of CRISPR/Cas9 systems targeting specific tissues and cells directly in vivo. In this Perspective, we focus on progress toward in vivo delivery of CRISPR/Cas components. Viral and nonviral delivery systems are both promising for gene editing in diverse tissues via local injection and systemic injection. We describe the various viral vectors and synthetic nonviral materials used for in vivo gene editing and applications to research and therapeutic models, and summarize opportunities and progress to date for both methods. We also discuss challenges for viral delivery, including overcoming limited packaging capacity, immunogenicity associated with multiple dosing, and the potential for off-target effects, and nonviral delivery, including efforts to increase efficacy and to expand utility of nonviral carriers for use in extrahepatic tissues and cancer. Looking ahead, additional advances in the safety and efficiency of viral and nonviral delivery systems for tissue- and cell-type-specific gene editing will be required to enable broad clinical translation. We provide a summary of current delivery systems used for in vivo genome editing, organized with respect to route of administration, and highlight immediate opportunities for biomedical research and applications. Furthermore, we discuss current challenges for in vivo delivery of CRISPR/Cas9 systems to guide the development of future therapies.
| S-EPMC7996671 | biostudies-literature
Project description:CRISPR/Cas, an adaptive immune system in bacteria, has been adopted as an efficient and precise tool for site-specific gene editing with potential therapeutic opportunities. It has been explored for a variety of applications, including gene modulation, epigenome editing, diagnosis, mRNA editing, etc. It has found applications in retinal dystrophic conditions including progressive cone and cone-rod dystrophies, congenital stationary night blindness, X-linked juvenile retinoschisis, retinitis pigmentosa, age-related macular degeneration, leber's congenital amaurosis, etc. Most of the therapies for retinal dystrophic conditions work by regressing symptoms instead of reversing the gene mutations. CRISPR/Cas9 through indel could impart beneficial effects in the reversal of gene mutations in dystrophic conditions. Recent research has also consolidated on the approaches of using CRISPR systems for retinal dystrophies but their delivery to the posterior part of the eye is a major concern due to high molecular weight, negative charge, and in vivo stability of CRISPR components. Recently, non-viral vectors have gained interest due to their potential in tissue-specific nucleic acid (miRNA/siRNA/CRISPR) delivery. This review highlights the opportunities of retinal dystrophies management using CRISPR/Cas nanomedicine.
| S-EPMC9614410 | biostudies-literature
Project description:CRISPR-Cas is an efficient method for genome editing in organisms from bacteria to human cells. We describe a transgene-free method for CRISPR-Cas-mediated cleavage in nematodes, enabling RNA-homology-targeted deletions that cause loss of gene function; analysis of whole-genome sequencing indicates that the nuclease activity is highly specific.
| S-EPMC3813845 | biostudies-other
Project description:In recent years, CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) genome editing systems have become one of the most robust platforms in basic biomedical research and therapeutic applications. To date, efficient in vivo delivery of the CRISPR/Cas9 system to the targeted cells remains a challenge. Although viral vectors have been widely used in the delivery of the CRISPR/Cas9 system in vitro and in vivo, their fundamental shortcomings, such as the risk of carcinogenesis, limited insertion size, immune responses and difficulty in large-scale production, severely limit their further applications. Alternative non-viral delivery systems for CRISPR/Cas9 are urgently needed. With the rapid development of non-viral vectors, lipid- or polymer-based nanocarriers have shown great potential for CRISPR/Cas9 delivery. In this review, we analyze the pros and cons of delivering CRISPR/Cas9 systems in the form of plasmid, mRNA, or protein and then discuss the limitations and challenges of CRISPR/Cas9-based genome editing. Furthermore, current non-viral vectors that have been applied for CRISPR/Cas9 delivery in vitro and in vivo are outlined in details. Finally, critical obstacles for non-viral delivery of CRISPR/Cas9 system are highlighted and promising strategies to overcome these barriers are proposed.
| S-EPMC5944364 | biostudies-literature
Project description:Genome editing in bacteria encompasses a wide array of laborious and multi-step methods such as suicide plasmids. The discovery and applications of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas based technologies have revolutionized genome editing in eukaryotic organisms due to its simplicity and programmability. Nevertheless, this system has not been as widely favored for bacterial genome editing. In this review, we summarize the main approaches and difficulties associated with CRISPR-Cas-mediated genome editing in bacteria and present some alternatives to circumvent these issues, including CRISPR nickases, Cas12a, base editors, CRISPR-associated transposases, prime-editing, endogenous CRISPR systems, and the use of pre-made ribonucleoprotein complexes of Cas proteins and guide RNAs. Finally, we also address fluorescent-protein-based methods to evaluate the efficacy of CRISPR-based systems for genome editing in bacteria. CRISPR-Cas still holds promise as a generalized genome-editing tool in bacteria and is developing further optimization for an expanded application in these organisms. This review provides a rarely offered comprehensive view of genome editing. It also aims to familiarize the microbiology community with an ever-growing genome-editing toolbox for bacteria.
| S-EPMC8071187 | biostudies-literature
Project description:The bacteria-derived CRISPR/Cas (an acronym for regularly interspaced short palindromic repeats/CRISPR-associated protein) system is currently the most widely used, versatile, and convenient tool for genome engineering. CRISPR/Cas-based technologies have been applied to disease modeling, gene therapies, transcriptional modulation, and diagnostics. Nevertheless, some challenges remain, such as the risk of immunological reactions or off-target effects. To overcome these problems, many new methods and CRISPR/Cas-based tools have been developed. In this review, we describe the current classification of CRISPR systems and new precise genome-editing technologies, summarize the latest applications of this technique in several fields of research, and, finally, discuss CRISPR/Cas system limitations, ethical issues, and challenges.
| S-EPMC10532350 | biostudies-literature
Project description:In the clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR associated protein (Cas) system, protoplasts are not only useful for rapidly validating the mutagenesis efficiency of various RNA-guided endonucleases, promoters, sgRNA designs, or Cas proteins, but can also be a platform for DNA-free gene editing. To date, the latter approach has been applied to numerous crops, particularly those with complex genomes, a long juvenile period, a tendency for heterosis, and/or self-incompatibility. Protoplast regeneration is thus a key step in DNA-free gene editing. In this report, we review the history and some future prospects for protoplast technology, including protoplast transfection, transformation, fusion, regeneration, and current protoplast applications in CRISPR/Cas-based breeding.
| S-EPMC8525356 | biostudies-literature
Project description:Genome-editing technology has emerged as a potential tool for treating incurable diseases for which few therapeutic modalities are available. In particular, discovery of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system together with the design of single-guide RNAs (sgRNAs) has sparked medical applications of genome editing. Despite the great promise of the CRISPR/Cas system, its clinical application is limited, in large part, by the lack of adequate delivery technology. To overcome this limitation, researchers have investigated various systems, including viral and nonviral vectors, for delivery of CRISPR/Cas and sgRNA into cells. Among nonviral delivery systems that have been studied are nanovesicles based on lipids, polymers, peptides, and extracellular vesicles. These nanovesicles have been designed to increase the delivery of CRISPR/Cas and sgRNA through endosome escape or using various stimuli such as light, pH, and environmental features. This review covers the latest research trends in nonviral, nanovesicle-based delivery systems that are being applied to genome-editing technology and suggests directions for future progress.
| S-EPMC7766488 | biostudies-literature
Project description:CRISPR system is a powerful defense mechanism in bacteria and archaea to provide immunity against viruses. Recently, this process found a new application in intended targeting of the genomes. CRISPR-mediated genome editing is performed by two main components namely single guide RNA and Cas9 protein. Despite the enormous data generated in this area, there is a dearth of high throughput resource. Therefore, we have developed CrisprGE, a central hub of CRISPR/Cas-based genome editing. Presently, this database holds a total of 4680 entries of 223 unique genes from 32 model and other organisms. It encompasses information about the organism, gene, target gene sequences, genetic modification, modifications length, genome editing efficiency, cell line, assay, etc. This depository is developed using the open source LAMP (Linux Apache MYSQL PHP) server. User-friendly browsing, searching facility is integrated for easy data retrieval. It also includes useful tools like BLAST CrisprGE, BLAST NTdb and CRISPR Mapper. Considering potential utilities of CRISPR in the vast area of biology and therapeutics, we foresee this platform as an assistance to accelerate research in the burgeoning field of genome engineering.
| S-EPMC4483309 | biostudies-literature
Project description:BackgroundThe true diploid frog, Xenopus tropicalis (X. tropicalis) is an excellent genetic model organism. To date, the CRISPR/Cas-mediated genome editing methods established in this species are mostly based on SpCas9 that requires the stringent NGG protospacer-adjacent motif (PAM) for target recognition, which limits its genome editing scope. Thus, it is highly desirable to circumvent this limitation.ResultsThrough one-cell stage injection of Cas/gRNAs into X. tropicalis embryos, we evaluated the mutagenic efficiency of 8 different Cas variants using T7EI assay, Sanger DNA sequencing, or deep sequencing. Our data indicate that SaCas9 and KKH SaCas9 are highly effective in frogs, which could be used for direct phenotyping in G0 embryos. In contrast, VQR Cas9, xCas9 3.7, SpG Cas9, and SpRY Cas9 were ineffective in X. tropicalis embryos and no activity was detected for iSpyMac Cas9. We also found that LbCas12a/crRNA RNP complexes with paired crRNAs efficiently induced small fragment deletions in X. tropicalis embryos.ConclusionSaCas9 and KKH SaCas9 are robust genome editing tools in X. tropicalis embryos. LbCas12a/crRNA RNP complexes are useful for inducing DNA fragment deletions in frog embryos. These tools expand the CRISPR/Cas genome editing scope in X. tropicalis and increase the flexibility for various genome editing applications in frogs.
| S-EPMC9264700 | biostudies-literature