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14 pages, 2027 KiB

Open AccessArticle

Delivery of CRISPR/Cas9 Plasmid DNA by Hyperbranched Polymeric Nanoparticles Enables Efficient Gene Editing

by Kemao Xiu, Laura Saunders, Luan Wen, Jinxue Ruan, Ruonan Dong, Jun Song, Dongshan Yang, Jifeng Zhang, Jie Xu, Y. Eugene Chen and Peter X. Ma

Cells 2023, 12(1), 156; https://doi.org/10.3390/cells12010156 - 30 Dec 2022

Cited by 7 | Viewed by 3926

Abstract

Gene editing nucleases such as CRISPR/Cas9 have enabled efficient and precise gene editing in vitro and hold promise of eventually achieving in vivo gene editing based therapy. However, a major challenge for their use is the lack of a safe and effective virus-free [...] Read more.

Gene editing nucleases such as CRISPR/Cas9 have enabled efficient and precise gene editing in vitro and hold promise of eventually achieving in vivo gene editing based therapy. However, a major challenge for their use is the lack of a safe and effective virus-free system to deliver gene editing nuclease elements. Polymers are a promising class of delivery vehicle due to their higher safety compared to currently used viral vectors, but polymers suffer from lower transfection efficiency. Polymeric vectors have been used for small nucleotide delivery but have yet to be used successfully with plasmid DNA (pDNA), which is often several hundred times larger than small nucleotides, presenting an engineering challenge. To address this, we extended our previously reported hyperbranched polymer (HP) delivery system for pDNA delivery by synthesizing several variants of HPs: HP-800, HP-1.8K, HP-10K, HP-25K. We demonstrate that all HPs have low toxicity in various cultured cells, with HP-25K being the most efficient at packaging and delivering pDNA. Importantly, HP-25K mediated delivery of CRISPR/Cas9 pDNA resulted in higher gene-editing rates than all other HPs and Lipofectamine at several clinically significant loci in different cell types. Consistently, HP-25K also led to more robust base editing when delivering the CRISPR base editor “BE4-max” pDNA to cells compared with Lipofectamine. The present work demonstrates that HP nanoparticles represent a promising class of vehicle for the non-viral delivery of pDNA towards the clinical application of gene-editing therapy. Full article

(This article belongs to the Special Issue CRISPR-Based Genome Editing in Translational Research)

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10 pages, 1989 KiB

Open AccessCommunication

Efficient and Safe Editing of Porcine Endogenous Retrovirus Genomes by Multiple-Site Base-Editing Editor

by Shuwen Zheng, Haiwen Zhong, Xiaoqing Zhou, Min Chen, Wansheng Li, Yin Zi, Yue Chi, Jinling Wang, Wei Zheng, Qingjian Zou, Liangxue Lai and Chengcheng Tang

Cells 2022, 11(24), 3975; https://doi.org/10.3390/cells11243975 - 8 Dec 2022

Cited by 3 | Viewed by 2324

Abstract

Gene-modified miniature pigs serve as alternative tissue and organ donors for xenotransplantation to alleviate the shortage of human allogenic organs. However, the high copy number of porcine endogenous retrovirus (PERV) genomes integrates with the porcine genome, which has a potential risk of cross-species [...] Read more.

Gene-modified miniature pigs serve as alternative tissue and organ donors for xenotransplantation to alleviate the shortage of human allogenic organs. However, the high copy number of porcine endogenous retrovirus (PERV) genomes integrates with the porcine genome, which has a potential risk of cross-species transmission and hinders the clinical practice of xenotransplantation. Recently, CRISPR/Cas9 has been used to inactivate PERVs. However, Cas9 also triggers severe DNA damage at multiple integrated PERV sites in the porcine genome, which induces senescence and apoptosis of porcine cells. In this study, the cytosine base editor (CBE), an efficient and safe editor that does not cause DNA double strand breaks (DSBs), was used for PERV editing to reduce cytotoxic effects. Seven sgRNAs were set to target gag and pol loci of PERVs to induce premature stop codons. We found that approximately 10% of cell clones were completely inactivated for PERVs in pig ST cells, and the plasmid that was used for editing the PERVs did not integrate into host genome and influence the karyotype of the modified cells. Our studies offer a powerful and safe strategy for further generating PERV-knockout pigs using base editors. Full article

(This article belongs to the Special Issue CRISPR-Based Genome Editing in Translational Research)

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15 pages, 2693 KiB

Open AccessArticle

Evaluating the Impact of gRNA SNPs in CasRx Activity for Reducing Viral RNA in HCoV-OC43

by Cathryn Michelle Mayes and Joshua Santarpia

Cells 2022, 11(12), 1859; https://doi.org/10.3390/cells11121859 - 7 Jun 2022

Cited by 2 | Viewed by 2432

Abstract

Viruses within a given family often share common essential genes that are highly conserved due to their critical role for the virus’s replication and survival. In this work, we developed a proof-of-concept for a pan-coronavirus CRISPR effector system by designing CRISPR targets that [...] Read more.

Viruses within a given family often share common essential genes that are highly conserved due to their critical role for the virus’s replication and survival. In this work, we developed a proof-of-concept for a pan-coronavirus CRISPR effector system by designing CRISPR targets that are cross-reactive among essential genes of different human coronaviruses (HCoV). We designed CRISPR targets for both the RNA-dependent RNA polymerase (RdRp) gene as well as the nucleocapsid (N) gene in coronaviruses. Using sequencing alignment, we determined the most highly conserved regions of these genes to design guide RNA (gRNA) sequences. In regions that were not completely homologous among HCoV species, we introduced mismatches into the gRNA sequence and tested the efficacy of CasRx, a Cas13d type CRISPR effector, using reverse transcription quantitative polymerase chain reaction (RT-qPCR) in HCoV-OC43. We evaluated the effect that mismatches in gRNA sequences has on the cleavage activity of CasRx and found that this CRISPR effector can tolerate up to three mismatches while still maintaining its nuclease activity in HCoV-OC43 viral RNA. This work highlights the need to evaluate off-target effects of CasRx with gRNAs containing up to three mismatches in order to design safe and effective CRISPR experiments. Full article

(This article belongs to the Special Issue CRISPR-Based Genome Editing in Translational Research)

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Review

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15 pages, 1594 KiB

Open AccessReview

Genome Editing for Cystic Fibrosis

by Guoshun Wang

Cells 2023, 12(12), 1555; https://doi.org/10.3390/cells12121555 - 6 Jun 2023

Cited by 16 | Viewed by 6057

Abstract

Cystic fibrosis (CF) is a monogenic recessive genetic disorder caused by mutations in the CF Transmembrane-conductance Regulator gene (CFTR). Remarkable progress in basic research has led to the discovery of highly effective CFTR modulators. Now ~90% of CF patients are treatable. [...] Read more.

Cystic fibrosis (CF) is a monogenic recessive genetic disorder caused by mutations in the CF Transmembrane-conductance Regulator gene (CFTR). Remarkable progress in basic research has led to the discovery of highly effective CFTR modulators. Now ~90% of CF patients are treatable. However, these modulator therapies are not curative and do not cover the full spectrum of CFTR mutations. Thus, there is a continued need to develop a complete and durable therapy that can treat all CF patients once and for all. As CF is a genetic disease, the ultimate therapy would be in-situ repair of the genetic lesions in the genome. Within the past few years, new technologies, such as CRISPR/Cas gene editing, have emerged as an appealing platform to revise the genome, ushering in a new era of genetic therapy. This review provided an update on this rapidly evolving field and the status of adapting the technology for CF therapy. Full article

(This article belongs to the Special Issue CRISPR-Based Genome Editing in Translational Research)

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39 pages, 2843 KiB

Open AccessReview

CRISPR-Cas System: The Current and Emerging Translational Landscape

by Narendranath Bhokisham, Ethan Laudermilch, Lindsay L. Traeger, Tonya D. Bonilla, Mercedes Ruiz-Estevez and Jordan R. Becker

Cells 2023, 12(8), 1103; https://doi.org/10.3390/cells12081103 - 7 Apr 2023

Cited by 16 | Viewed by 15576

Abstract

CRISPR-Cas technology has rapidly changed life science research and human medicine. The ability to add, remove, or edit human DNA sequences has transformative potential for treating congenital and acquired human diseases. The timely maturation of the cell and gene therapy ecosystem and its [...] Read more.

CRISPR-Cas technology has rapidly changed life science research and human medicine. The ability to add, remove, or edit human DNA sequences has transformative potential for treating congenital and acquired human diseases. The timely maturation of the cell and gene therapy ecosystem and its seamless integration with CRISPR-Cas technologies has enabled the development of therapies that could potentially cure not only monogenic diseases such as sickle cell anemia and muscular dystrophy, but also complex heterogenous diseases such as cancer and diabetes. Here, we review the current landscape of clinical trials involving the use of various CRISPR-Cas systems as therapeutics for human diseases, discuss challenges, and explore new CRISPR-Cas-based tools such as base editing, prime editing, CRISPR-based transcriptional regulation, CRISPR-based epigenome editing, and RNA editing, each promising new functionality and broadening therapeutic potential. Finally, we discuss how the CRISPR-Cas system is being used to understand the biology of human diseases through the generation of large animal disease models used for preclinical testing of emerging therapeutics. Full article

(This article belongs to the Special Issue CRISPR-Based Genome Editing in Translational Research)

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24 pages, 1837 KiB

Open AccessReview

Future Perspectives of Prime Editing for the Treatment of Inherited Retinal Diseases

by Silja Hansen, Michelle E. McClements, Thomas J. Corydon and Robert E. MacLaren

Cells 2023, 12(3), 440; https://doi.org/10.3390/cells12030440 - 29 Jan 2023

Cited by 16 | Viewed by 6903

Abstract

Inherited retinal diseases (IRD) are a clinically and genetically heterogenous group of diseases and a leading cause of blindness in the working-age population. Even though gene augmentation therapies have shown promising results, they are only feasible to treat a small number of autosomal [...] Read more.

Inherited retinal diseases (IRD) are a clinically and genetically heterogenous group of diseases and a leading cause of blindness in the working-age population. Even though gene augmentation therapies have shown promising results, they are only feasible to treat a small number of autosomal recessive IRDs, because the size of the gene is limited by the vector used. DNA editing however could potentially correct errors regardless of the overall size of the gene and might also be used to correct dominant mutations. Prime editing is a novel CRISPR/Cas9 based gene editing tool that enables precise correction of point mutations, insertions, and deletions without causing double strand DNA breaks. Due to its versatility and precision this technology may be a potential treatment option for virtually all genetic causes of IRD. Since its initial description, the prime editing technology has been further improved, resulting in higher efficacy and a larger target scope. Additionally, progress has been achieved concerning the size-related delivery issue of the prime editor components. This review aims to give an overview of these recent advancements and discusses prime editing as a potential treatment for IRDs. Full article

(This article belongs to the Special Issue CRISPR-Based Genome Editing in Translational Research)

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32 pages, 6930 KiB

Open AccessReview

CRISPR-Cas9 Technology for the Creation of Biological Avatars Capable of Modeling and Treating Pathologies: From Discovery to the Latest Improvements

by Ali Nasrallah, Eric Sulpice, Farah Kobaisi, Xavier Gidrol and Walid Rachidi

Cells 2022, 11(22), 3615; https://doi.org/10.3390/cells11223615 - 15 Nov 2022

Cited by 8 | Viewed by 7887

Abstract

This is a spectacular moment for genetics to evolve in genome editing, which encompasses the precise alteration of the cellular DNA sequences within various species. One of the most fascinating genome-editing technologies currently available is Clustered Regularly Interspaced Palindromic Repeats (CRISPR) and its [...] Read more.

This is a spectacular moment for genetics to evolve in genome editing, which encompasses the precise alteration of the cellular DNA sequences within various species. One of the most fascinating genome-editing technologies currently available is Clustered Regularly Interspaced Palindromic Repeats (CRISPR) and its associated protein 9 (CRISPR-Cas9), which have integrated deeply into the research field within a short period due to its effectiveness. It became a standard tool utilized in a broad spectrum of biological and therapeutic applications. Furthermore, reliable disease models are required to improve the quality of healthcare. CRISPR-Cas9 has the potential to diversify our knowledge in genetics by generating cellular models, which can mimic various human diseases to better understand the disease consequences and develop new treatments. Precision in genome editing offered by CRISPR-Cas9 is now paving the way for gene therapy to expand in clinical trials to treat several genetic diseases in a wide range of species. This review article will discuss genome-editing tools: CRISPR-Cas9, Zinc Finger Nucleases (ZFNs), and Transcription Activator-Like Effector Nucleases (TALENs). It will also encompass the importance of CRISPR-Cas9 technology in generating cellular disease models for novel therapeutics, its applications in gene therapy, and challenges with novel strategies to enhance its specificity. Full article

(This article belongs to the Special Issue CRISPR-Based Genome Editing in Translational Research)

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26 pages, 1459 KiB

Open AccessReview

CRISPR-Based Therapeutic Gene Editing for Duchenne Muscular Dystrophy: Advances, Challenges and Perspectives

by Guofang Chen, Tingyi Wei, Hui Yang, Guoling Li and Haisen Li

Cells 2022, 11(19), 2964; https://doi.org/10.3390/cells11192964 - 22 Sep 2022

Cited by 16 | Viewed by 7396

Abstract

Duchenne muscular dystrophy (DMD) is a severe neuromuscular disease arising from loss-of-function mutations in the dystrophin gene and characterized by progressive muscle degeneration, respiratory insufficiency, cardiac failure, and premature death by the age of thirty. Albeit DMD is one of the most common [...] Read more.

Duchenne muscular dystrophy (DMD) is a severe neuromuscular disease arising from loss-of-function mutations in the dystrophin gene and characterized by progressive muscle degeneration, respiratory insufficiency, cardiac failure, and premature death by the age of thirty. Albeit DMD is one of the most common types of fatal genetic diseases, there is no curative treatment for this devastating disorder. In recent years, gene editing via the clustered regularly interspaced short palindromic repeats (CRISPR) system has paved a new path toward correcting pathological mutations at the genetic source, thus enabling the permanent restoration of dystrophin expression and function throughout the musculature. To date, the therapeutic benefits of CRISPR genome-editing systems have been successfully demonstrated in human cells, rodents, canines, and piglets with diverse DMD mutations. Nevertheless, there remain some nonignorable challenges to be solved before the clinical application of CRISPR-based gene therapy. Herein, we provide an overview of therapeutic CRISPR genome-editing systems, summarize recent advancements in their applications in DMD contexts, and discuss several potential obstacles lying ahead of clinical translation. Full article

(This article belongs to the Special Issue CRISPR-Based Genome Editing in Translational Research)

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15 pages, 1386 KiB

Open AccessEditor’s ChoiceReview

Recent Advances in Improving Gene-Editing Specificity through CRISPR–Cas9 Nuclease Engineering

by Xiaoqiang Huang, Dongshan Yang, Jifeng Zhang, Jie Xu and Y. Eugene Chen

Cells 2022, 11(14), 2186; https://doi.org/10.3390/cells11142186 - 13 Jul 2022

Cited by 32 | Viewed by 6746

Abstract

CRISPR–Cas9 is the state-of-the-art programmable genome-editing tool widely used in many areas. For safe therapeutic applications in clinical medicine, its off-target effect must be dramatically minimized. In recent years, extensive studies have been conducted to improve the gene-editing specificity of the most popular [...] Read more.

CRISPR–Cas9 is the state-of-the-art programmable genome-editing tool widely used in many areas. For safe therapeutic applications in clinical medicine, its off-target effect must be dramatically minimized. In recent years, extensive studies have been conducted to improve the gene-editing specificity of the most popular CRISPR–Cas9 nucleases using different strategies. In this review, we summarize and discuss these strategies and achievements, with a major focus on improving the gene-editing specificity through Cas9 protein engineering. Full article

(This article belongs to the Special Issue CRISPR-Based Genome Editing in Translational Research)

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