Journal articles: 'Correction génique (CRISPR/Cas9)'

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Jordan, Bertrand. "CRISPR-Cas9, une nouvelle donne pour la thérapie génique." médecine/sciences 31, no. 11 (November 2015): 1035–38. http://dx.doi.org/10.1051/medsci/20153111018.

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Cheng, Hao, Feng Zhang, and Yang Ding. "CRISPR/Cas9 Delivery System Engineering for Genome Editing in Therapeutic Applications." Pharmaceutics 13, no. 10 (October 9, 2021): 1649. http://dx.doi.org/10.3390/pharmaceutics13101649.

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The clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (CRISPR/Cas9) systems have emerged as a robust and versatile genome editing platform for gene correction, transcriptional regulation, disease modeling, and nucleic acids imaging. However, the insufficient transfection and off-target risks have seriously hampered the potential biomedical applications of CRISPR/Cas9 technology. Herein, we review the recent progress towards CRISPR/Cas9 system delivery based on viral and non-viral vectors. We summarize the CRISPR/Cas9-inspired clinical trials and analyze the CRISPR/Cas9 delivery technology applied in the trials. The rational-designed non-viral vectors for delivering three typical forms of CRISPR/Cas9 system, including plasmid DNA (pDNA), mRNA, and ribonucleoprotein (RNP, Cas9 protein complexed with gRNA) were highlighted in this review. The vector-derived strategies to tackle the off-target concerns were further discussed. Moreover, we consider the challenges and prospects to realize the clinical potential of CRISPR/Cas9-based genome editing.

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Yun, Yeomin, and Yoon Ha. "CRISPR/Cas9-Mediated Gene Correction to Understand ALS." International Journal of Molecular Sciences 21, no. 11 (May 27, 2020): 3801. http://dx.doi.org/10.3390/ijms21113801.

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Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease caused by the death of motor neurons in the spinal cord and brainstem. ALS has a diverse genetic origin; at least 20 genes have been shown to be related to ALS. Most familial and sporadic cases of ALS are caused by variants of the SOD1, C9orf72, FUS, and TARDBP genes. Genome editing using clustered regularly interspaced short palindromic repeats/CRISPR-associated system 9 (CRISPR/Cas9) can provide insights into the underlying genetics and pathophysiology of ALS. By correcting common mutations associated with ALS in animal models and patient-derived induced pluripotent stem cells (iPSCs), CRISPR/Cas9 has been used to verify the effects of ALS-associated mutations and observe phenotype differences between patient-derived and gene-corrected iPSCs. This technology has also been used to create mutations to investigate the pathophysiology of ALS. Here, we review recent studies that have used CRISPR/Cas9 to understand the genetic underpinnings of ALS.

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Walther, Johanna, Danny Wilbie, Vincent S. J. Tissingh, Mert Öktem, Heleen van der Veen, Bo Lou, and Enrico Mastrobattista. "Impact of Formulation Conditions on Lipid Nanoparticle Characteristics and Functional Delivery of CRISPR RNP for Gene Knock-Out and Correction." Pharmaceutics 14, no. 1 (January 17, 2022): 213. http://dx.doi.org/10.3390/pharmaceutics14010213.

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The CRISPR-Cas9 system is an emerging therapeutic tool with the potential to correct diverse genetic disorders. However, for gene therapy applications, an efficient delivery vehicle is required, capable of delivering the CRISPR-Cas9 components into the cytosol of the intended target cell population. In this study, we optimized the formulation conditions of lipid nanoparticles (LNP) for delivery of ready-made CRISPR-Cas9 ribonucleic protein (RNP). The buffer composition during complexation and relative DOTAP concentrations were varied for LNP encapsulating in-house produced Cas9 RNP alone or Cas9 RNP with additional template DNA for gene correction. The LNP were characterized for size, surface charge, and plasma interaction through asymmetric flow field flow fractionation (AF4). Particles were functionally screened on fluorescent reporter cell lines for gene knock-out and gene correction. This revealed incompatibility of RNP with citrate buffer and PBS. We demonstrated that LNP for gene knock-out did not necessarily require DOTAP, while LNP for gene correction were only active with a low concentration of DOTAP. The AF4 studies additionally revealed that LNP interact with plasma, however, remain stable, whereby HDR template seems to favor stability of LNP. Under optimal formulation conditions, we achieved gene knock-out and gene correction efficiencies as high as 80% and 20%, respectively, at nanomolar concentrations of the CRISPR-Cas9 RNP.

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Men, Ke, Xingmei Duan, Zhiyao He, Yang Yang, Shaohua Yao, and Yuquan Wei. "CRISPR/Cas9-mediated correction of human genetic disease." Science China Life Sciences 60, no. 5 (May 2017): 447–57. http://dx.doi.org/10.1007/s11427-017-9032-4.

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Hainzl, S., P. Peking, T. Kocher, E. M. Murauer, F. Larcher, M. del Río, B. G. Duarte, et al. "185 CRISPR/Cas9 mediated gene correction of COL7A1." Journal of Investigative Dermatology 137, no. 10 (October 2017): S224. http://dx.doi.org/10.1016/j.jid.2017.07.182.

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Hanafy, Amira Sayed, Susanne Schoch, and Alf Lamprecht. "CRISPR/Cas9 Delivery Potentials in Alzheimer’s Disease Management: A Mini Review." Pharmaceutics 12, no. 9 (August 25, 2020): 801. http://dx.doi.org/10.3390/pharmaceutics12090801.

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Alzheimer’s disease (AD) is the most common dementia disorder. While genetic mutations account for only 1% of AD cases, sporadic AD resulting from a combination of genetic and risk factors constitutes >90% of the cases. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein (Cas9) is an impactful gene editing tool which identifies a targeted gene sequence, creating a double-stranded break followed by gene inactivation or correction. Although CRISPR/Cas9 can be utilized to irreversibly inactivate or correct faulty genes in AD, a safe and effective delivery system stands as a challenge against the translation of CRISPR therapeutics from bench to bedside. While viral vectors are efficient in CRISPR/Cas9 delivery, they might introduce fatal side effects and immune responses. As non-viral vectors offer a better safety profile, cost-effectiveness and versatility, they can be promising for the in vivo delivery of CRISPR/Cas9 therapeutics. In this minireview, we present an overview of viral and non-viral vector based CRISPR/Cas9 therapeutic strategies that are being evaluated on pre-clinical AD models. Other promising non-viral vectors that can be used for genome editing in AD, such as nanoparticles, nanoclews and microvesicles, are also discussed. Finally, we list the formulation and technical aspects that must be considered in order to develop a successful non-viral CRISPR/Cas9 delivery vehicle.

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Jo, Dong Hyun, Dong Woo Song, Chang Sik Cho, Un Gi Kim, Kyu Jun Lee, Kihwang Lee, Sung Wook Park, et al. "CRISPR-Cas9–mediated therapeutic editing of Rpe65 ameliorates the disease phenotypes in a mouse model of Leber congenital amaurosis." Science Advances 5, no. 10 (October 2019): eaax1210. http://dx.doi.org/10.1126/sciadv.aax1210.

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Leber congenital amaurosis (LCA), one of the leading causes of childhood-onset blindness, is caused by autosomal recessive mutations in several genes including RPE65. In this study, we performed CRISPR-Cas9–mediated therapeutic correction of a disease-associated nonsense mutation in Rpe65 in rd12 mice, a model of human LCA. Subretinal injection of adeno-associated virus carrying CRISPR-Cas9 and donor DNA resulted in >1% homology-directed repair and ~1.6% deletion of the pathogenic stop codon in Rpe65 in retinal pigment epithelial tissues of rd12 mice. The a- and b-waves of electroretinograms were recovered to levels up to 21.2 ± 4.1% and 39.8 ± 3.2% of their wild-type mice counterparts upon bright stimuli after dark adaptation 7 months after injection. There was no definite evidence of histologic perturbation or tumorigenesis during 7 months of observation. Collectively, we present the first therapeutic correction of an Rpe65 nonsense mutation using CRISPR-Cas9, providing new insight for developing therapeutics for LCA.

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Atmanli, Ayhan, Andreas C. Chai, Miao Cui, Zhaoning Wang, Takahiko Nishiyama, Rhonda Bassel-Duby, and Eric N. Olson. "Cardiac Myoediting Attenuates Cardiac Abnormalities in Human and Mouse Models of Duchenne Muscular Dystrophy." Circulation Research 129, no. 6 (September 3, 2021): 602–16. http://dx.doi.org/10.1161/circresaha.121.319579.

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Rationale: Absence of dystrophin in Duchenne muscular dystrophy (DMD) results in the degeneration of skeletal and cardiac muscles. Owing to advances in respiratory management of patients with DMD, cardiomyopathy has become a significant aspect of the disease. While CRISPR/Cas9 genome editing technology holds great potential as a novel therapeutic avenue for DMD, little is known about the potential of DMD correction using CRISPR/Cas9 technology to mitigate cardiac abnormalities in DMD. Objective: To define the effects of CRISPR/Cas9 genome editing on structural, functional, and transcriptional abnormalities in DMD-associated cardiac disease. Methods and Results: We generated induced pluripotent stem cells from a patient with a deletion of exon 44 of the DMD gene (ΔEx44) and his healthy brother. We targeted exon 45 of the DMD gene by CRISPR/Cas9 genome editing to generate corrected DMD induced pluripotent stem cell lines, wherein the DMD open reading frame was restored via reframing or exon skipping. While DMD cardiomyocytes demonstrated morphological, structural, and functional deficits compared with control cardiomyocytes, cardiomyocytes from both corrected DMD lines were similar to control cardiomyocytes. Bulk RNA-sequencing of DMD cardiomyocytes showed transcriptional dysregulation consistent with dilated cardiomyopathy, which was mitigated in corrected DMD cardiomyocytes. We then corrected dysfunctional DMD cardiomyocytes by adenoviral delivery of Cas9/gRNA and showed that correction of DMD cardiomyocytes postdifferentiation reduces their arrhythmogenic potential. Single-nucleus RNA-sequencing of hearts of DMD mice showed transcriptional dysregulation in cardiomyocytes and fibroblasts, which in corrected mice was reduced to similar levels as wild-type mice. Conclusions: We show that CRISPR/Cas9-mediated correction of DMD ΔEx44 mitigates structural, functional, and transcriptional abnormalities consistent with dilated cardiomyopathy irrespective of how the protein reading frame is restored. We show that these effects extend to postnatal editing in induced pluripotent stem cell-derived cardiomyocytes and mice. These findings provide key insights into the utility of genome editing as a novel therapeutic for DMD-associated cardiomyopathy.

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Luo, Yumei, Detu Zhu, Zhizhuo Zhang, Yaoyong Chen, and Xiaofang Sun. "Integrative Analysis of CRISPR/Cas9 Target Sites in the HumanHBBGene." BioMed Research International 2015 (2015): 1–9. http://dx.doi.org/10.1155/2015/514709.

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Recently, the clustered regularly interspaced short palindromic repeats (CRISPR) system has emerged as a powerful customizable artificial nuclease to facilitate precise genetic correction for tissue regeneration and isogenic disease modeling. However, previous studies reported substantial off-target activities of CRISPR system in human cells, and the enormous putative off-target sites are labor-intensive to be validated experimentally, thus motivating bioinformatics methods for rational design of CRISPR system and prediction of its potential off-target effects. Here, we describe an integrative analytical process to identify specific CRISPR target sites in the humanβ-globin gene (HBB) and predict their off-target effects. Our method includes off-target analysis in both coding and noncoding regions, which was neglected by previous studies. It was found that the CRISPR target sites in the introns have fewer off-target sites in the coding regions than those in the exons. Remarkably, target sites containing certain transcriptional factor motif have enriched binding sites of relevant transcriptional factor in their off-target sets. We also found that the intron sites have fewer SNPs, which leads to less variation of CRISPR efficiency in different individuals during clinical applications. Our studies provide a standard analytical procedure to select specific CRISPR targets for genetic correction.

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Pöhler, Michael, Sarah Guttmann, Oksana Nadzemova, Malte Lenders, Eva Brand, Andree Zibert, Hartmut H. Schmidt, and Vanessa Sandfort. "CRISPR/Cas9-mediated correction of mutated copper transporter ATP7B." PLOS ONE 15, no. 9 (September 30, 2020): e0239411. http://dx.doi.org/10.1371/journal.pone.0239411.

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Xia, Emily, Rongqi Duan, Fushan Shi, Kyle E. Seigel, Hartmut Grasemann, and Jim Hu. "Overcoming the Undesirable CRISPR-Cas9 Expression in Gene Correction." Molecular Therapy - Nucleic Acids 13 (December 2018): 699–709. http://dx.doi.org/10.1016/j.omtn.2018.10.015.

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Zhang, Yu, Hui Li, Yi-Li Min, Efrain Sanchez-Ortiz, Jian Huang, Alex A. Mireault, John M. Shelton, et al. "Enhanced CRISPR-Cas9 correction of Duchenne muscular dystrophy in mice by a self-complementary AAV delivery system." Science Advances 6, no. 8 (February 2020): eaay6812. http://dx.doi.org/10.1126/sciadv.aay6812.

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Duchenne muscular dystrophy (DMD) is a lethal neuromuscular disease caused by mutations in the dystrophin gene (DMD). Previously, we applied CRISPR-Cas9–mediated “single-cut” genome editing to correct diverse genetic mutations in animal models of DMD. However, high doses of adeno-associated virus (AAV) are required for efficient in vivo genome editing, posing challenges for clinical application. In this study, we packaged Cas9 nuclease in single-stranded AAV (ssAAV) and CRISPR single guide RNAs in self-complementary AAV (scAAV) and delivered this dual AAV system into a mouse model of DMD. The dose of scAAV required for efficient genome editing were at least 20-fold lower than with ssAAV. Mice receiving systemic treatment showed restoration of dystrophin expression and improved muscle contractility. These findings show that the efficiency of CRISPR-Cas9–mediated genome editing can be substantially improved by using the scAAV system. This represents an important advancement toward therapeutic translation of genome editing for DMD.

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Jinka, Chaitra. "CRISPR-Cas9 gene editing and human diseases." Bioinformation 18, no. 11 (November 30, 2022): 1081–86. http://dx.doi.org/10.6026/973206300181081.

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CRISPR/Cas-9 mediated genome editing has recently emerged as a potential and innovative technology in therapeutic development and biomedical research. Several recent studies have been performed to understand gene modification techniques in obtaining effective ex vivo results. Generally, the disease targets for gene correction will be in specific organs, so understanding the complete potential of genomic treatment methods is essential. From such a perspective, the present review revealed the significant importance of the CRISPR/ Cas9 delivery system. Both the promising gene-editing delivery systems, such as synthetic (non-viral) and viral vector systems are discussed in this review. In addition, this paper attempted to summarize the tissue-specific and organ-specific mRNA delivery systems that provide possible research information for future researchers. Further, the major challenges of the CRISPR/Cas9 system, such as off-target delivery, immunogenicity, and limited packaging, were also elucidated. Accordingly, this review illustrated a wide range of clinical applications associated with the efficient delivery of CRISPR/ Cas9 gene-editing. Moreover, this article emphasizes the role of the CRISPR/Cas9 system in treating Intra Cerebral haemorrhage (ICH), thereby suggesting future researchers to adopt more clinical trials on this breakthrough delivery system.

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Santos, Renato, and Olga Amaral. "Advances in Sphingolipidoses: CRISPR-Cas9 Editing as an Option for Modelling and Therapy." International Journal of Molecular Sciences 20, no. 23 (November 24, 2019): 5897. http://dx.doi.org/10.3390/ijms20235897.

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Sphingolipidoses are inherited genetic diseases characterized by the accumulation of glycosphingolipids. Sphingolipidoses (SP), which usually involve the loss of sphingolipid hydrolase function, are of lysosomal origin, and represent an important group of rare diseases among lysosomal storage disorders. Initial treatments consisted of enzyme replacement therapy, but, in recent decades, various therapeutic approaches have been developed. However, these commonly used treatments for SP fail to be fully effective and do not penetrate the blood–brain barrier. New approaches, such as genome editing, have great potential for both the treatment and study of sphingolipidoses. Here, we review the most recent advances in the treatment and modelling of SP through the application of CRISPR-Cas9 genome editing. CRISPR-Cas9 is currently the most widely used method for genome editing. This technique is versatile; it can be used for altering the regulation of genes involved in sphingolipid degradation and synthesis pathways, interrogating gene function, generating knock out models, or knocking in mutations. CRISPR-Cas9 genome editing is being used as an approach to disease treatment, but more frequently it is utilized to create models of disease. New CRISPR-Cas9-based tools of gene editing with diminished off-targeting effects are evolving and seem to be more promising for the correction of individual mutations. Emerging Prime results and CRISPR-Cas9 difficulties are also discussed.

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Charpentier, Emmanuelle. "Gene Editing and Genome Engineering with CRISPR-Cas9." Molecular Frontiers Journal 01, no. 02 (December 2017): 99–107. http://dx.doi.org/10.1142/s2529732517400119.

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The RNA-programmable CRISPR-Cas9 technology allows precise and efficient engineering or correction of mutations, modulation of gene expression and marking of DNA in a wide variety of cell types and organisms in the three domains of life. Because of its versatility and ease of design, this powerful technology has been rapidly and universally adopted for genome editing applications in life science research. It is also recognized for its promising and potentially transformative applications in biotechnology, medicine and agriculture.

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Walsh, Colin, and Sha Jin. "Induced Pluripotent Stem Cells and CRISPR-Cas9 Innovations for Treating Alpha-1 Antitrypsin Deficiency and Glycogen Storage Diseases." Cells 13, no. 12 (June 18, 2024): 1052. http://dx.doi.org/10.3390/cells13121052.

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Human induced pluripotent stem cell (iPSC) and CRISPR-Cas9 gene-editing technologies have become powerful tools in disease modeling and treatment. By harnessing recent biotechnological advancements, this review aims to equip researchers and clinicians with a comprehensive and updated understanding of the evolving treatment landscape for metabolic and genetic disorders, highlighting how iPSCs provide a unique platform for detailed pathological modeling and pharmacological testing, driving forward precision medicine and drug discovery. Concurrently, CRISPR-Cas9 offers unprecedented precision in gene correction, presenting potential curative therapies that move beyond symptomatic treatment. Therefore, this review examines the transformative role of iPSC technology and CRISPR-Cas9 gene editing in addressing metabolic and genetic disorders such as alpha-1 antitrypsin deficiency (A1AD) and glycogen storage disease (GSD), which significantly impact liver and pulmonary health and pose substantial challenges in clinical management. In addition, this review discusses significant achievements alongside persistent challenges such as technical limitations, ethical concerns, and regulatory hurdles. Future directions, including innovations in gene-editing accuracy and therapeutic delivery systems, are emphasized for next-generation therapies that leverage the full potential of iPSC and CRISPR-Cas9 technologies.

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Amoasii, Leonela, Chengzu Long, Hui Li, Alex A. Mireault, John M. Shelton, Efrain Sanchez-Ortiz, John R. McAnally, et al. "Single-cut genome editing restores dystrophin expression in a new mouse model of muscular dystrophy." Science Translational Medicine 9, no. 418 (November 29, 2017): eaan8081. http://dx.doi.org/10.1126/scitranslmed.aan8081.

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Duchenne muscular dystrophy (DMD) is a severe, progressive muscle disease caused by mutations in the dystrophin gene. The majority of DMD mutations are deletions that prematurely terminate the dystrophin protein. Deletions of exon 50 of the dystrophin gene are among the most common single exon deletions causing DMD. Such mutations can be corrected by skipping exon 51, thereby restoring the dystrophin reading frame. Using clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9), we generated a DMD mouse model by deleting exon 50. These ΔEx50 mice displayed severe muscle dysfunction, which was corrected by systemic delivery of adeno-associated virus encoding CRISPR/Cas9 genome editing components. We optimized the method for dystrophin reading frame correction using a single guide RNA that created reframing mutations and allowed skipping of exon 51. In conjunction with muscle-specific expression of Cas9, this approach restored up to 90% of dystrophin protein expression throughout skeletal muscles and the heart of ΔEx50 mice. This method of permanently bypassing DMD mutations using a single cut in genomic DNA represents a step toward clinical correction of DMD mutations and potentially those of other neuromuscular disorders.

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Bravo, Jack P. K., Mu-Sen Liu, Grace N. Hibshman, Tyler L. Dangerfield, Kyungseok Jung, Ryan S. McCool, Kenneth A. Johnson, and David W. Taylor. "Publisher Correction: Structural basis for mismatch surveillance by CRISPR–Cas9." Nature 604, no. 7904 (March 22, 2022): E10. http://dx.doi.org/10.1038/s41586-022-04655-8.

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Schaefer, Kellie A., Wen-Hsuan Wu, Diana F. Colgan, Stephen H. Tsang, Alexander G. Bassuk, and Vinit B. Mahajan. "Correction: Retraction: Unexpected mutations after CRISPR–Cas9 editing in vivo." Nature Methods 15, no. 5 (May 2018): 394. http://dx.doi.org/10.1038/nmeth0518-394a.

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Kawashima, Nozomu, Yusuke Okuno, Yuko Sekiya, Xinan Wang, Atsushi Narita, Sayoko Doisaki, Michi Kamei, et al. "Correction of Fanconi Anemia Mutation Using the Crispr/Cas9 System." Blood 126, no. 23 (December 3, 2015): 3622. http://dx.doi.org/10.1182/blood.v126.23.3622.3622.

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Abstract Introduction Gene therapy has been developed for genetic diseases, either to restore normal function for loss-of-function mutations or to inhibit gain-of-function mutations. Gene addition using genetically engineered viral and plasmid vectors has successfully corrected cell pathophysiology resulting in the production of functional proteins. Therapeutic safety has been reinforced by the use of self-inactivating vectors; however, the potential risk of tumorigenesis raises concerns for insertional mutagenesis combined with acquired somatic mutations. Recent advances in gene editing using an RNA-guided endonuclease (RGEN), known as the CRISPR/Cas9 system, have opened a new frontier for the in situ correction of disease-associated mutations. Genomic DNA of cells harboring mutations can be excised and replaced with a DNA template for the functional gene sequence using homology-directed repair (HDR). The advantages of this repair include fewer off-target effects and a reduced risk of copy number changes compared with gene addition using vectors. Fanconi anemia (FA) is a syndrome of inherited bone marrow failure, characterized by the deficient regulation of DNA double-strand break repair. Clinical trials of gene therapy using viral vectors are still on-going with partial success; therefore, a new gene editing technique deserves attention. However, the feasibility of this approach in diseases with impaired HDR, such as FA, is unknown. Therefore, we used an RGEN to generate a cell line harboring a disease-causing point mutation in an FA-associated gene and elucidated the efficacy of restoring the mutation thereafter. Methods pSpCas9(BB) (PX330) was used to express humanized S. pyogenes Cas9 and single guide RNAs (sgRNAs) of interest. The sgRNAs were designed by searching for NGG protospacer adjacent motif (PAM) sequences near the point mutation target sites. The candidate sgRNAs were designed to be specific for the FANCC c.67delG:p.D23Ifs*23 mutation type (MT) or wild type (WT): gRNA#4, 5′-ATGGGATCAGGCTTCCACTT-3′ and gRNA#5, 5′-GAAGCTTTCTGTATGGGATC-3′ were specific for the WT sequence; whereas, gRNA M4, 5′-TATGGATCAGGCTTCCACTT-3′ and gRNA M5, 5′-AGAAGCTTTCTGTATGGATC-3′ were specific for the MT sequence. pCAG-EGxxFP, an EGFP-based reporter plasmid for the HDR that harbored the 500-bp target region of the WT or MT FANCC, was constructed for the gRNA selection. An HDR template construct was designed to incorporate a puromycin-resistant gene flanked by two loxP sites and two homologous arms containing the WT or MT sequence. HEK293T cells harboring the WT FANCC sequence were genetically edited by the above-mentioned plasmids. Results To validate an efficient and specific sgRNA for DNA double-strand breaks, we cotransfected pCAG-EGxxFP-FANCC WT or MT and pSpCas9(BB)-FANCC-gRNA plasmids into HEK293T cells. EGFP fluorescence, whose intensity is correlated with the efficacy of HDR and thus the efficacy and specificity of sequence-specific DNA excision, was observed 48 h later, and we determined that gRNA#4 and gRNA M4 were specific for the WT and MT sequences, respectively. To generate cells harboring the MT FANCC sequence, HEK293T cells were cotransfected with pSpCas9(BB)-FANCC-gRNA#4 and the HDR template plasmid harboring the MT FANCC. A cell harboring biallelic MT FANCC was selected by adding puromycin and single-cell cloning. The transient expression of Cre recombinase in this clone successfully deleted the drug-selection cassette, and 293T-FANCC c.67delG cells were established. This cell showed the loss of FANCD2 monoubiquitination, a hallmark of a deficient FA core complex. Next, the 293T-FANCC c.67delG cells were cotransfected with pSpCas9(BB)-FANCC-gRNA M4 and the HDR template with the WT FANCC. This restoration of the mutated FANCC sequence resulted in a high frequency of at least monoallelic correction and the restoration of FANCD2 monoubiquitination. Conclusions The feasibility of genome editing was demonstrated in cells harboring an FA mutation, which can be a foothold for future therapy using precision gene restoration in patients with impaired HDR. Disclosures No relevant conflicts of interest to declare.

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Li, Dandan, Minglin Ou, Wei Zhang, Qi Luo, Wanxia Cai, Chune Mo, Wenken Liang, et al. "CRISPR/Cas9-Mediated Gene Correction in Osteopetrosis Patient-Derived iPSCs." Frontiers in Bioscience-Landmark 28, no. 6 (June 30, 2023): 131. http://dx.doi.org/10.31083/j.fbl2806131.

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Wade, Mark. "High-Throughput Silencing Using the CRISPR-Cas9 System." Journal of Biomolecular Screening 20, no. 8 (May 22, 2015): 1027–39. http://dx.doi.org/10.1177/1087057115587916.

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The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system has been seized upon with a fervor enjoyed previously by small interfering RNA (siRNA) and short hairpin RNA (shRNA) technologies and has enormous potential for high-throughput functional genomics studies. The decision to use this approach must be balanced with respect to adoption of existing platforms versus awaiting the development of more “mature” next-generation systems. Here, experience from siRNA and shRNA screening plays an important role, as issues such as targeting efficiency, pooling strategies, and off-target effects with those technologies are already framing debates in the CRISPR field. CRISPR/Cas can be exploited not only to knockout genes but also to up- or down-regulate gene transcription—in some cases in a multiplex fashion. This provides a powerful tool for studying the interaction among multiple signaling cascades in the same genetic background. Furthermore, the documented success of CRISPR/Cas-mediated gene correction (or the corollary, introduction of disease-specific mutations) provides proof of concept for the rapid generation of isogenic cell lines for high-throughput screening. In this review, the advantages and limitations of CRISPR/Cas are discussed and current and future applications are highlighted. It is envisaged that complementarities between CRISPR, siRNA, and shRNA will ensure that all three technologies remain critical to the success of future functional genomics projects.

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Padayachee, Jananee, and Moganavelli Singh. "Therapeutic applications of CRISPR/Cas9 in breast cancer and delivery potential of gold nanomaterials." Nanobiomedicine 7 (January 1, 2020): 184954352098319. http://dx.doi.org/10.1177/1849543520983196.

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Globally, approximately 1 in 4 cancers in women are diagnosed as breast cancer (BC). Despite significant advances in the diagnosis and therapy BCs, many patients develop metastases or relapses. Hence, novel therapeutic strategies are required, that can selectively and efficiently kill malignant cells. Direct targeting of the genetic and epigenetic aberrations that occur in BC development is a promising strategy to overcome the limitations of current therapies, which target the tumour phenotype. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system, composed of only an easily modifiable single guide RNA (sgRNA) sequence bound to a Cas9 nuclease, has revolutionised genome editing due to its simplicity and efficiency compared to earlier systems. CRISPR/Cas9 and its associated catalytically inactivated dCas9 variants facilitate the knockout of overexpressed genes, correction of mutations in inactivated genes, and reprogramming of the epigenetic landscape to impair BC growth. To achieve efficient genome editing in vivo, a vector is required to deliver the components to target cells. Gold nanomaterials, including gold nanoparticles and nanoclusters, display many advantageous characteristics that have facilitated their widespread use in theranostics, as delivery vehicles, and imaging and photothermal agents. This review highlights the therapeutic applications of CRISPR/Cas9 in treating BCs, and briefly describes gold nanomaterials and their potential in CRISPR/Cas9 delivery.

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Beretta, Maxime, and Hugo Mouquet. "Ingénierie de lymphocytes B humains produisant des anticorps neutralisant le virus VIH-1 par édition génique CRISPR-Cas9." médecine/sciences 35, no. 12 (December 2019): 993–96. http://dx.doi.org/10.1051/medsci/2019196.

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Les anticorps (ou immunoglobulines, Ig) produits par les lymphocytes B sont essentiels aux réponses immunitaires induites par les infections et les vaccins. Les anticorps sont des glycoprotéines hétérodimériques résultant de l’association de deux chaînes lourdes (IgH), et de deux chaînes légères (IgL) d’immunoglobuline. Les chaînes IgH et IgL possèdent des régions « hypervariables », également appelées en anglais complementarity determining regions (CDR), situées dans leurs domaines variables, VH et VL, qui, en se combinant, forment le site de liaison à l’antigène ou paratope.

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Han, Xin, Zongbin Liu, Myeong chan Jo, Kai Zhang, Ying Li, Zihua Zeng, Nan Li, Youli Zu, and Lidong Qin. "CRISPR-Cas9 delivery to hard-to-transfect cells via membrane deformation." Science Advances 1, no. 7 (August 2015): e1500454. http://dx.doi.org/10.1126/sciadv.1500454.

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The CRISPR (clustered regularly interspaced short palindromic repeats)–Cas (CRISPR-associated) nuclease system represents an efficient tool for genome editing and gene function analysis. It consists of two components: single-guide RNA (sgRNA) and the enzyme Cas9. Typical sgRNA and Cas9 intracellular delivery techniques are limited by their reliance on cell type and exogenous materials as well as their toxic effects on cells (for example, electroporation). We introduce and optimize a microfluidic membrane deformation method to deliver sgRNA and Cas9 into different cell types and achieve successful genome editing. This approach uses rapid cell mechanical deformation to generate transient membrane holes to enable delivery of biomaterials in the medium. We achieved high delivery efficiency of different macromolecules into different cell types, including hard-to-transfect lymphoma cells and embryonic stem cells, while maintaining high cell viability. With the advantages of broad applicability across different cell types, particularly hard-to-transfect cells, and flexibility of application, this method could potentially enable new avenues of biomedical research and gene targeting therapy such as mutation correction of disease genes through combination of the CRISPR-Cas9–mediated knockin system.

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Jung, Hyerin, Yeri Alice Rim, Narae Park, Yoojun Nam, and Ji Hyeon Ju. "Restoration of Osteogenesis by CRISPR/Cas9 Genome Editing of the Mutated COL1A1 Gene in Osteogenesis Imperfecta." Journal of Clinical Medicine 10, no. 14 (July 16, 2021): 3141. http://dx.doi.org/10.3390/jcm10143141.

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Osteogenesis imperfecta (OI) is a genetic disease characterized by bone fragility and repeated fractures. The bone fragility associated with OI is caused by a defect in collagen formation due to mutation of COL1A1 or COL1A2. Current strategies for treating OI are not curative. In this study, we generated induced pluripotent stem cells (iPSCs) from OI patient-derived blood cells harboring a mutation in the COL1A1 gene. Osteoblast (OB) differentiated from OI-iPSCs showed abnormally decreased levels of type I collagen and osteogenic differentiation ability. Gene correction of the COL1A1 gene using CRISPR/Cas9 recovered the decreased type I collagen expression in OBs differentiated from OI-iPSCs. The osteogenic potential of OI-iPSCs was also recovered by the gene correction. This study suggests a new possibility of treatment and in vitro disease modeling using patient-derived iPSCs and gene editing with CRISPR/Cas9.

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Miki, Toshio, Ludivina Vazquez, Lisa Yanuaria, Omar Lopez, Irving M. Garcia, Kazuo Ohashi, and Natalie S. Rodriguez. "Induced Pluripotent Stem Cell Derivation and Ex Vivo Gene Correction Using a Mucopolysaccharidosis Type 1 Disease Mouse Model." Stem Cells International 2019 (April 1, 2019): 1–10. http://dx.doi.org/10.1155/2019/6978303.

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Mucopolysaccharidosis type 1 (MPS-1), also known as Hurler’s disease, is a congenital metabolic disorder caused by a mutation in the alpha-L-iduronidase (IDUA) gene, which results in the loss of lysosomal enzyme function for the degradation of glycosaminoglycans. Here, we demonstrate the proof of concept of ex vivo gene editing therapy using induced pluripotent stem cell (iPSC) and CRISPR/Cas9 technologies with MPS-1 model mouse cell. Disease-affected iPSCs were generated from Idua knockout mouse embryonic fibroblasts, which carry a disrupting neomycin-resistant gene cassette (Neor) in exon VI of the Idua gene. Double guide RNAs were used to remove the Neor sequence, and various lengths of donor templates were used to reconstruct the exon VI sequence. A quantitative PCR-based screening method was used to identify Neor removal. The sequence restoration without any indel mutation was further confirmed by Sanger sequencing. After induced fibroblast differentiation, the gene-corrected iPSC-derived fibroblasts demonstrated Idua function equivalent to the wild-type iPSC-derived fibroblasts. The Idua-deficient cells were competent to be reprogrammed to iPSCs, and pluripotency was maintained through CRISPR/CAS9-mediated gene correction. These results support the concept of ex vivo gene editing therapy using iPSC and CRISPR/Cas9 technologies for MPS-1 patients.

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Vibhuti Choubisa and Sunil Sharma. "Unveiling neural network potential in forecasting CRISPR effects and off-target prophecies for gene editing." International Journal of Science and Research Archive 10, no. 1 (September 30, 2023): 252–59. http://dx.doi.org/10.30574/ijsra.2023.10.1.0738.

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The revolutionary Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) - associated protein 9 (Cas9) systems has emerged as a groundbreaking gene-editing tool, widely embraced within biomedical research. Nonetheless, the utilization of guide RiboNucleic Acids (gRNAs) in the CRISPR-Cas9 system can inadvertently trigger undesired off-target effects, consequently impinging on the practical implementation of this technique. Existing in silico prediction methods that focus on off-target effects have exhibited constrained predictive accuracy, necessitating further enhancement. To tackle this challenge, we present a Base Editing and Prime Editing approach in this study. This approach aim to enhance the precision and specificity of DeoxyRiboNucleic Acid (DNA) modifications compared to traditional CRISPR-Cas9 methods. Both techniques provide unique approaches to achieve targeted changes in the DNA sequence without inducing double-stranded breaks, which can lead to off-target effects. Base editing is highly specific and allows for the correction of point mutations associated with diseases while Prime Editing allows for a wider range of modifications compared to base editing, including the ability to insert or delete specific sequences. Their ability to achieve specific genetic changes while minimizing off-target effects makes them valuable additions to the gene editing toolkit. The findings of this research contribute to the advancement of precision gene editing, offering an enhanced predictive framework to mitigate off-target effects in the realm of CRISPR-Cas9 technology.

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Joung, Julia, Silvana Konermann, Jonathan S. Gootenberg, Omar O. Abudayyeh, Randall J. Platt, Mark D. Brigham, Neville E. Sanjana, and Feng Zhang. "Author Correction: Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening." Nature Protocols 14, no. 7 (October 22, 2018): 2259. http://dx.doi.org/10.1038/s41596-018-0063-0.

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Morishige, Satoshi, Shinichi Mizuno, Hidetoshi Ozawa, Takayuki Nakamura, Ahmad Mazahery, Kei Nomura, Ritsuko Seki, et al. "CRISPR/Cas9-mediated gene correction in hemophilia B patient-derived iPSCs." International Journal of Hematology 111, no. 2 (October 29, 2019): 225–33. http://dx.doi.org/10.1007/s12185-019-02765-0.

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Alanis-Lobato, Gregorio, Jasmin Zohren, Afshan McCarthy, Norah M. E. Fogarty, Nada Kubikova, Emily Hardman, Maria Greco, Dagan Wells, James M. A. Turner, and Kathy K. Niakan. "Frequent loss of heterozygosity in CRISPR-Cas9–edited early human embryos." Proceedings of the National Academy of Sciences 118, no. 22 (April 9, 2021): e2004832117. http://dx.doi.org/10.1073/pnas.2004832117.

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CRISPR-Cas9 genome editing is a promising technique for clinical applications, such as the correction of disease-associated alleles in somatic cells. The use of this approach has also been discussed in the context of heritable editing of the human germ line. However, studies assessing gene correction in early human embryos report low efficiency of mutation repair, high rates of mosaicism, and the possibility of unintended editing outcomes that may have pathologic consequences. We developed computational pipelines to assess single-cell genomics and transcriptomics datasets from OCT4 (POU5F1) CRISPR-Cas9–targeted and control human preimplantation embryos. This allowed us to evaluate on-target mutations that would be missed by more conventional genotyping techniques. We observed loss of heterozygosity in edited cells that spanned regions beyond the POU5F1 on-target locus, as well as segmental loss and gain of chromosome 6, on which the POU5F1 gene is located. Unintended genome editing outcomes were present in ∼16% of the human embryo cells analyzed and spanned 4–20 kb. Our observations are consistent with recent findings indicating complexity at on-target sites following CRISPR-Cas9 genome editing. Our work underscores the importance of further basic research to assess the safety of genome editing techniques in human embryos, which will inform debates about the potential clinical use of this technology.

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Cosenza, Lucia Carmela, Cristina Zuccato, Matteo Zurlo, Roberto Gambari, and Alessia Finotti. "Co-Treatment of Erythroid Cells from β-Thalassemia Patients with CRISPR-Cas9-Based β039-Globin Gene Editing and Induction of Fetal Hemoglobin." Genes 13, no. 10 (September 26, 2022): 1727. http://dx.doi.org/10.3390/genes13101727.

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Gene editing (GE) is an efficient strategy for correcting genetic mutations in monogenic hereditary diseases, including β-thalassemia. We have elsewhere reported that CRISPR-Cas9-based gene editing can be employed for the efficient correction of the β039-thalassemia mutation. On the other hand, robust evidence demonstrates that the increased production of fetal hemoglobin (HbF) can be beneficial for patients with β-thalassemia. The aim of our study was to verify whether the de novo production of adult hemoglobin (HbA) using CRISPR-Cas9 gene editing can be combined with HbF induction protocols. The gene editing of the β039-globin mutation was obtained using a CRISPR-Cas9-based experimental strategy; the correction of the gene sequence and the transcription of the corrected gene were analyzed by allele-specific droplet digital PCR and RT-qPCR, respectively; the relative content of HbA and HbF was studied by high-performance liquid chromatography (HPLC) and Western blotting. For HbF induction, the repurposed drug rapamycin was used. The data obtained conclusively demonstrate that the maximal production of HbA and HbF is obtained in GE-corrected, rapamycin-induced erythroid progenitors isolated from β039-thalassemia patients. In conclusion, GE and HbF induction might be used in combination in order to achieve the de novo production of HbA together with an increase in induced HbF.

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Chung, Sun-Ku, and Seo-Young Lee. "Advances in Gene Therapy Techniques to Treat LRRK2 Gene Mutation." Biomolecules 12, no. 12 (December 5, 2022): 1814. http://dx.doi.org/10.3390/biom12121814.

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Leucine-rich repeat kinase 2 (LRRK2) gene mutation is an autosomal dominant mutation associated with Parkinson’s disease (PD). Among LRRK2 gene mutations, the LRRK2 G2019S mutation is frequently involved in PD onset. Currently, diverse gene correction tools such as zinc finger nucleases (ZFNs), helper-dependent adenoviral vector (HDAdV), the bacterial artificial chromosome-based homologous recombination (BAC-based HR) system, and CRISPR/Cas9-homology-directed repair (HDR) or adenine base editor (ABE) are used in genome editing. Gene correction of the LRRK2 G2019S mutation has been applied whenever new gene therapy tools emerge, being mainly applied to induced pluripotent stem cells (LRRK2 G2019S-mutant iPSCs). Here, we comprehensively introduce the principles and methods of each programmable nuclease such as ZFN, CRISPR/Cas9-HDR or ABE applied to LRRK2 G2019S, as well as those of HDAdV or BAC-based HR systems used as nonprogrammable nuclease systems.

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Ababneh, Nidaa A., Jakub Scaber, Rowan Flynn, Andrew Douglas, Paola Barbagallo, Ana Candalija, Martin R. Turner, et al. "Correction of amyotrophic lateral sclerosis related phenotypes in induced pluripotent stem cell-derived motor neurons carrying a hexanucleotide expansion mutation in C9orf72 by CRISPR/Cas9 genome editing using homology-directed repair." Human Molecular Genetics 29, no. 13 (June 5, 2020): 2200–2217. http://dx.doi.org/10.1093/hmg/ddaa106.

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Abstract The G4C2 hexanucleotide repeat expansion (HRE) in C9orf72 is the commonest cause of familial amyotrophic lateral sclerosis (ALS). A number of different methods have been used to generate isogenic control lines using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and non-homologous end-joining by deleting the repeat region, with the risk of creating indels and genomic instability. In this study, we demonstrate complete correction of an induced pluripotent stem cell (iPSC) line derived from a C9orf72-HRE positive ALS/frontotemporal dementia patient using CRISPR/Cas9 genome editing and homology-directed repair (HDR), resulting in replacement of the excised region with a donor template carrying the wild-type repeat size to maintain the genetic architecture of the locus. The isogenic correction of the C9orf72 HRE restored normal gene expression and methylation at the C9orf72 locus, reduced intron retention in the edited lines and abolished pathological phenotypes associated with the C9orf72 HRE expansion in iPSC-derived motor neurons (iPSMNs). RNA sequencing of the mutant line identified 2220 differentially expressed genes compared with its isogenic control. Enrichment analysis demonstrated an over-representation of ALS relevant pathways, including calcium ion dependent exocytosis, synaptic transport and the Kyoto Encyclopedia of Genes and Genomes ALS pathway, as well as new targets of potential relevance to ALS pathophysiology. Complete correction of the C9orf72 HRE in iPSMNs by CRISPR/Cas9-mediated HDR provides an ideal model to study the earliest effects of the hexanucleotide expansion on cellular homeostasis and the key pathways implicated in ALS pathophysiology.

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Pavani, Giulia, Anna Fabiano, Marine Laurent, Fatima Amor, Erika Cantelli, Anne Chalumeau, Giulia Maule, et al. "Correction of β-thalassemia by CRISPR/Cas9 editing of the α-globin locus in human hematopoietic stem cells." Blood Advances 5, no. 5 (February 26, 2021): 1137–53. http://dx.doi.org/10.1182/bloodadvances.2020001996.

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Abstract β-thalassemias (β-thal) are a group of blood disorders caused by mutations in the β-globin gene (HBB) cluster. β-globin associates with α-globin to form adult hemoglobin (HbA, α2β2), the main oxygen-carrier in erythrocytes. When β-globin chains are absent or limiting, free α-globins precipitate and damage cell membranes, causing hemolysis and ineffective erythropoiesis. Clinical data show that severity of β-thal correlates with the number of inherited α-globin genes (HBA1 and HBA2), with α-globin gene deletions having a beneficial effect for patients. Here, we describe a novel strategy to treat β-thal based on genome editing of the α-globin locus in human hematopoietic stem/progenitor cells (HSPCs). Using CRISPR/Cas9, we combined 2 therapeutic approaches: (1) α-globin downregulation, by deleting the HBA2 gene to recreate an α-thalassemia trait, and (2) β-globin expression, by targeted integration of a β-globin transgene downstream the HBA2 promoter. First, we optimized the CRISPR/Cas9 strategy and corrected the pathological phenotype in a cellular model of β-thalassemia (human erythroid progenitor cell [HUDEP-2] β0). Then, we edited healthy donor HSPCs and demonstrated that they maintained long-term repopulation capacity and multipotency in xenotransplanted mice. To assess the clinical potential of this approach, we next edited β-thal HSPCs and achieved correction of α/β globin imbalance in HSPC-derived erythroblasts. As a safer option for clinical translation, we performed editing in HSPCs using Cas9 nickase showing precise editing with no InDels. Overall, we described an innovative CRISPR/Cas9 approach to improve α/β globin imbalance in thalassemic HSPCs, paving the way for novel therapeutic strategies for β-thal.

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Chen, Chiao-Lin, Jonathan Rodiger, Verena Chung, Raghuvir Viswanatha, Stephanie E. Mohr, Yanhui Hu, and Norbert Perrimon. "SNP-CRISPR: A Web Tool for SNP-Specific Genome Editing." G3: Genes|Genomes|Genetics 10, no. 2 (December 10, 2019): 489–94. http://dx.doi.org/10.1534/g3.119.400904.

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CRISPR-Cas9 is a powerful genome editing technology in which a single guide RNA (sgRNA) confers target site specificity to achieve Cas9-mediated genome editing. Numerous sgRNA design tools have been developed based on reference genomes for humans and model organisms. However, existing resources are not optimal as genetic mutations or single nucleotide polymorphisms (SNPs) within the targeting region affect the efficiency of CRISPR-based approaches by interfering with guide-target complementarity. To facilitate identification of sgRNAs (1) in non-reference genomes, (2) across varying genetic backgrounds, or (3) for specific targeting of SNP-containing alleles, for example, disease relevant mutations, we developed a web tool, SNP-CRISPR (https://www.flyrnai.org/tools/snp_crispr/). SNP-CRISPR can be used to design sgRNAs based on public variant data sets or user-identified variants. In addition, the tool computes efficiency and specificity scores for sgRNA designs targeting both the variant and the reference. Moreover, SNP-CRISPR provides the option to upload multiple SNPs and target single or multiple nearby base changes simultaneously with a single sgRNA design. Given these capabilities, SNP-CRISPR has a wide range of potential research applications in model systems and for design of sgRNAs for disease-associated variant correction.

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Susani, Lucia, Alessandra Castelli, Michela Lizier, Franco Lucchini, Paolo Vezzoni, and Marianna Paulis. "Correction of a Recessive Genetic Defect by CRISPR-Cas9-Mediated Endogenous Repair." CRISPR Journal 1, no. 3 (June 2018): 230–38. http://dx.doi.org/10.1089/crispr.2018.0004.

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Öktem, Mert, Enrico Mastrobattista, and Olivier G. de Jong. "Amphipathic Cell-Penetrating Peptide-Aided Delivery of Cas9 RNP for In Vitro Gene Editing and Correction." Pharmaceutics 15, no. 10 (October 20, 2023): 2500. http://dx.doi.org/10.3390/pharmaceutics15102500.

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The therapeutic potential of the CRISPR-Cas9 gene editing system in treating numerous genetic disorders is immense. To fully realize this potential, it is crucial to achieve safe and efficient delivery of CRISPR-Cas9 components into the nuclei of target cells. In this study, we investigated the applicability of the amphipathic cell-penetrating peptide LAH5, previously employed for DNA delivery, in the intracellular delivery of spCas9:sgRNA ribonucleoprotein (RNP) and the RNP/single-stranded homology-directed repair (HDR) template. Our findings reveal that the LAH5 peptide effectively formed nanocomplexes with both RNP and RNP/HDR cargo, and these nanocomplexes demonstrated successful cellular uptake and cargo delivery. The loading of all RNP/HDR components into LAH5 nanocomplexes was confirmed using an electrophoretic mobility shift assay. Functional screening of various ratios of peptide/RNP nanocomplexes was performed on fluorescent reporter cell lines to assess gene editing and HDR-mediated gene correction. Moreover, targeted gene editing of the CCR5 gene was successfully demonstrated across diverse cell lines. This LAH5-based delivery strategy represents a significant advancement toward the development of therapeutic delivery systems for CRISPR-Cas-based genetic engineering in in vitro and ex vivo applications.

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Min, Yi-Li, Hui Li, Cristina Rodriguez-Caycedo, Alex A. Mireault, Jian Huang, John M. Shelton, John R. McAnally, et al. "CRISPR-Cas9 corrects Duchenne muscular dystrophy exon 44 deletion mutations in mice and human cells." Science Advances 5, no. 3 (March 2019): eaav4324. http://dx.doi.org/10.1126/sciadv.aav4324.

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Mutations in the dystrophin gene cause Duchenne muscular dystrophy (DMD), which is characterized by lethal degeneration of cardiac and skeletal muscles. Mutations that delete exon 44 of the dystrophin gene represent one of the most common causes of DMD and can be corrected in ~12% of patients by editing surrounding exons, which restores the dystrophin open reading frame. Here, we present a simple and efficient strategy for correction of exon 44 deletion mutations by CRISPR-Cas9 gene editing in cardiomyocytes obtained from patient-derived induced pluripotent stem cells and in a new mouse model harboring the same deletion mutation. Using AAV9 encoding Cas9 and single guide RNAs, we also demonstrate the importance of the dosages of these gene editing components for optimal gene correction in vivo. Our findings represent a significant step toward possible clinical application of gene editing for correction of DMD.

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Schnütgen, Frank, Duran Sürün, Joachim Schwäble, Ana Tomasovic, Ralf Kühn, Stefan Stein, Nina Kurrle, Hubert Serve, Erhard Seifried, and Harald von Melchner. "High Efficiency Gene Correction in Hematopoietic Cells By Template-Free Crispr/Cas9 Genome Editing." Blood 128, no. 22 (December 2, 2016): 3507. http://dx.doi.org/10.1182/blood.v128.22.3507.3507.

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Abstract Introduction A significant fraction of inherited monogenic disorders is caused by patient-specific mutations dispersed over the entire locus of the affected gene. Correcting these mutations by introducing healthy gene copies into the genome of the diseased cells proved successful in several clinical gene therapy trials. Most of these trials employed retroviral vectors, which by inserting randomly throughout the genome deprived the transduced genes of their endogenous control and caused insertional mutations leading to secondary disease. The development of genome editing tools capable of modifying any prespecified genomic sequence with unprecedented accuracy opened up a wide range of new possibilities in gene manipulation including targeted gene repair. In particular, CRISPR/Cas9, a prokaryotic adaptive immune system, and its swift repurposing for genome editing was widely adopted as the hitherto simplest genome editing tool. CRISPR/Cas9 is an RNA guided endonuclease that uses RNA-DNA base pairing to target genomic DNA. Bound to its target via the guide (g)RNA, Cas9 induces DNA double strand breaks (DBS) at prespecified genomic sites that promptly activate the endogenous DNA repair machinery. DSB repair is accomplished by either non-homologous end joining (NHEJ) or homology directed repair (HDR). Thus far, correction of human mutations in hematopoietic cells relied entirely on HDR requiring gene specific donor templates in addition to the RNA guided endonucleases (RGNs). However, although the HDR offers precision, its efficiency is low and requires positive selection to enrich for gene corrected cell. Because in mammalian cells DSB repair by NHEJ significantly exceeds HDR and even more importantly, is the dominant DSB repair pathway in hematopoietic stem- and progenitor cells (HSPC), we thought to exploit NHEJ for gene therapy because in theory, approximately one third of the indels associated with NHEJ should restore the open reading frame (ORF) disrupted by a disease mutation. This would lead to a significant number of ORF reconstitutions of which some, depending on the position and type of the original mutation, should either completely or partially recover protein function. Results To test gene repair efficiency by NHEJ in human hematopoietic cells, we generated PLB-985 (PLB) reporter cells expressing mutationally inactivated EGFP (mEGFP). Transduction of mEGFP expressing PLBs (mEGFP-PLB) with integrase-deficient lentiviral (IDLV) particles encoding for RGNs targeting the EGFP mutation reconstituted EGFP expression in up to 27% of the mEGFP PLB cells. Indel analysis revealed that 13 out of 28 (46%) restored the EGFP-open reading frame of which 7 (25%) reconstituted EGFP activity. To test whether the donor-template free IDLV strategy would also effectively correct bone fide disease mutations, we performed similar experiments with X-CGD PLB cells expressing transgenes encoding patient-specific frameshift, missense and nonsense CYBB mutations causing X-linked chronic granulomatous disease (X-CGD) which is an inherited, life threatening immunodeficiency disorder. Transduction of the cells with IDLVs carrying RGNs directed against each of these mutations restored CYBB function in up to 10% of cells harboring frameshift mutations which is sufficient to protect X-CGD patients from microbial infections. However, RGNs directed against the nonsense or missense mutations restored CYBB function in only 1-2% of the cells, suggesting that these mutations are less amenable to CRISPR/Cas9 mediated repair. As Cas9 frequently tolerates single nucleotide mismatches, selection against solitary nucleotide substitutions may explain this low efficiency of gene repair. Conclusions Frameshift mutations can be effectively repaired by NHEJ in hematopoietic cells by CRISPR/Cas9 transducing IDLVs. As about 25% of most inherited blood disorders are caused by frameshift mutations, our results suggest that about a quarter of patients suffering from monogenic blood disorders could benefit from personalized, template free CRISPR/Cas9 gene therapy. Disclosures No relevant conflicts of interest to declare.

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Gobalakrishnan, Krishshan, Vignesh Jayarajan, Veronica Kinsler, and Wei-Li Di. "O18 Precision genome editing for targeted correction of pathogenic D50N mutation in keratitis–ichthyosis–deafness syndrome using CRISPR/Cas9 and homology-directed repair." British Journal of Dermatology 190, no. 6 (May 17, 2024): e76-e77. http://dx.doi.org/10.1093/bjd/ljae105.018.

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Abstract Introduction and aims Keratitis–ichthyosis–deafness (KID) syndrome is an ectodermal disorder that causes blindness, skin inflammation and deafness. It is caused by autosomal dominant mutations in the gap junction beta 2 (GJB2) gene with 86% being a hotspot mutation on c.148G>A, resulting in the amino acid replacement (D50N) in its coding protein connexin 26 (Cx26). There is currently no curative treatment for KID syndrome. We aim to correct the hotspot mutation D50N using a genome-editing approach. Methods Keratinocytes generated from the patient with KID who had a D50N mutation (KID-KCs) were genetically corrected by homologous-directed repair/CRISPR-Cas9 using an electroporation approach. The correction efficiency was determined and analysed using Sanger sequencing and Synthego Inference of CRISPR Edits analysis. The off-target effects were confirmed by whole-exome sequencing [exome next-generation sequencing (NGS)]. Furthermore, the functional recovery following genome editing was assessed by GJB2 mRNA production, Cx26 protein expression, and trafficking and hemichannel activity. Results The correction efficiencies ranged from 95% to 100% with different doses of CRISPR-Cas9 and 8µg CRISPR-Cas9 was the most optimal concentration with the efficiency of 100% and approximately 0% insertion-deletion (INDEL). Fifty days after gene editing, the INDEL increased to only 2%, indicating a minimum residual CRISPR-Cas9 effect in edited cells. The exome-NGS analysis also showed no off-target effects in the outside target area. Functional studies showed that there was a significant increase in wildtype GJB2 mRNA expression and a reduction of mutant GJB2 expression (n = 5, P < 0.01). Increased Cx26 membranous localization in KID-KCs was also observed (n = 3, P < 0.01). Additionally, Cx26 hemichannel activity assessment using the neurobiotin assay showed a significant decrease in ‘leaky’ hemichannel in gene-edited KID-KCs compared with unedited cells (n = 3, P < 0.05). Conclusions We can genome-correct KID keratinocytes harbouring the mutation D50N with 100% editing efficiency, 0–2% INDEL, and undetectable off-target effects. This result indicates a promising gene therapy for KID syndrome with the hotspot mutation D50N.

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Bohrer, Laura, Luke Wiley, Erin Burnight, Jessica Cooke, Joseph Giacalone, Kristin Anfinson, Jeaneen Andorf, Robert Mullins, Edwin Stone, and Budd Tucker. "Correction of NR2E3 Associated Enhanced S-cone Syndrome Patient-specific iPSCs using CRISPR-Cas9." Genes 10, no. 4 (April 5, 2019): 278. http://dx.doi.org/10.3390/genes10040278.

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Enhanced S-cone syndrome (ESCS) is caused by recessive mutations in the photoreceptor cell transcription factor NR2E3. Loss of NR2E3 is characterized by repression of rod photoreceptor cell gene expression, over-expansion of the S-cone photoreceptor cell population, and varying degrees of M- and L-cone photoreceptor cell development. In this study, we developed a CRISPR-based homology-directed repair strategy and corrected two different disease-causing NR2E3 mutations in patient-derived induced pluripotent stem cells (iPSCs) generated from two affected individuals. In addition, one patient’s iPSCs were differentiated into retinal cells and NR2E3 transcription was evaluated in CRISPR corrected and uncorrected clones. The patient’s c.119-2A>C mutation caused the inclusion of a portion of intron 1, the creation of a frame shift, and generation of a premature stop codon. In summary, we used a single set of CRISPR reagents to correct different mutations in iPSCs generated from two individuals with ESCS. In doing so we demonstrate the advantage of using retinal cells derived from affected patients over artificial in vitro model systems when attempting to demonstrate pathophysiologic mechanisms of specific mutations.

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Sürün, Duran, Aksana Schneider, Jovan Mircetic, Katrin Neumann, Felix Lansing, Maciej Paszkowski-Rogacz, Vanessa Hänchen, Min Ae Lee-Kirsch, and Frank Buchholz. "Efficient Generation and Correction of Mutations in Human iPS Cells Utilizing mRNAs of CRISPR Base Editors and Prime Editors." Genes 11, no. 5 (May 6, 2020): 511. http://dx.doi.org/10.3390/genes11050511.

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In contrast to CRISPR/Cas9 nucleases, CRISPR base editors (BE) and prime editors (PE) enable predefined nucleotide exchanges in genomic sequences without generating DNA double strand breaks. Here, we employed BE and PE mRNAs in conjunction with chemically synthesized sgRNAs and pegRNAs for efficient editing of human induced pluripotent stem cells (iPSC). Whereas we were unable to correct a disease-causing mutation in patient derived iPSCs using a CRISPR/Cas9 nuclease approach, we corrected the mutation back to wild type with high efficiency utilizing an adenine BE. We also used adenine and cytosine BEs to introduce nine different cancer associated TP53 mutations into human iPSCs with up to 90% efficiency, generating a panel of cell lines to investigate the biology of these mutations in an isogenic background. Finally, we pioneered the use of prime editing in human iPSCs, opening this important cell type for the precise modification of nucleotides not addressable by BEs and to multiple nucleotide exchanges. These approaches eliminate the necessity of deriving disease specific iPSCs from human donors and allows the comparison of different disease-causing mutations in isogenic genetic backgrounds.

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Skvarova Kramarzova, Karolina, Mark Osborn, Beau Webber, Anthony DeFeo, Amber McElroy, Chong Kim, and Jakub Tolar. "CRISPR/Cas9-Mediated Correction of the FANCD1 Gene in Primary Patient Cells." International Journal of Molecular Sciences 18, no. 6 (June 14, 2017): 1269. http://dx.doi.org/10.3390/ijms18061269.

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Hoban, Megan D., Dianne Lumaquin, Caroline Y. Kuo, Zulema Romero, Joseph Long, Michelle Ho, Courtney S. Young, et al. "CRISPR/Cas9-Mediated Correction of the Sickle Mutation in Human CD34+ cells." Molecular Therapy 24, no. 9 (September 2016): 1561–69. http://dx.doi.org/10.1038/mt.2016.148.

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Wu, Yuxuan, Dan Liang, Yinghua Wang, Meizhu Bai, Wei Tang, Shiming Bao, Zhiqiang Yan, Dangsheng Li, and Jinsong Li. "Correction of a Genetic Disease in Mouse via Use of CRISPR-Cas9." Cell Stem Cell 13, no. 6 (December 2013): 659–62. http://dx.doi.org/10.1016/j.stem.2013.10.016.

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Li, Hongmei Lisa, Peter Gee, Kentaro Ishida, and Akitsu Hotta. "Efficient genomic correction methods in human iPS cells using CRISPR–Cas9 system." Methods 101 (May 2016): 27–35. http://dx.doi.org/10.1016/j.ymeth.2015.10.015.

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Smirnikhina, S., A. Anuchina, E. Adilgereeva, K. Kochergin-Nikitsky, and A. Lavrov. "WS09.3 Development of effective method for F508del mutation correction using CRISPR/Cas9." Journal of Cystic Fibrosis 17 (June 2018): S17. http://dx.doi.org/10.1016/s1569-1993(18)30169-3.

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Yingjun, Xie, Xie Yuhuan, Chen Yuchang, Li Dongzhi, Wang Ding, Song Bing, Yang Yi, et al. "CRISPR/Cas9 gene correction of HbH-CS thalassemia-induced pluripotent stem cells." Annals of Hematology 98, no. 12 (September 9, 2019): 2661–71. http://dx.doi.org/10.1007/s00277-019-03763-2.

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Journal articles on the topic 'Correction génique (CRISPR/Cas9)'

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