Advances in therapeutic application of CRISPR-Cas9

2019 ◽  
Vol 19 (3) ◽  
pp. 164-174 ◽  
Author(s):  
Jinyu Sun ◽  
Jianchu Wang ◽  
Donghui Zheng ◽  
Xiaorong Hu
Keyword(s):  
Gene Therapy ◽  
Genome Editing ◽  
Malignant Tumor ◽  
Gene Editing ◽  
Genome Engineering ◽  
Therapeutic Application ◽  
Adaptive Immune ◽  
Efficient Gene

Abstract Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) is one of the most versatile and efficient gene editing technologies, which is derived from adaptive immune strategies for bacteria and archaea. With the remarkable development of programmable nuclease-based genome engineering these years, CRISPR-Cas9 system has developed quickly in recent 5 years and has been widely applied in countless areas, including genome editing, gene function investigation and gene therapy both in vitro and in vivo. In this paper, we briefly introduce the mechanisms of CRISPR-Cas9 tool in genome editing. More importantly, we review the recent therapeutic application of CRISPR-Cas9 in various diseases, including hematologic diseases, infectious diseases and malignant tumor. Finally, we discuss the current challenges and consider thoughtfully what advances are required in order to further develop the therapeutic application of CRISPR-Cas9 in the future.

scholarly journals Comprehensive deletion landscape of CRISPR-Cas9 identifies minimal RNA-guided DNA-binding modules

2020 ◽  
Author(s):  
Arik Shams ◽  
Sean A. Higgins ◽  
Christof Fellmann ◽  
Thomas G. Laughlin ◽  
Benjamin L. Oakes ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Dna Binding ◽  
Genome Editing ◽  
Experimental Validation ◽  
Effector Proteins ◽  
Multidomain Proteins ◽  
General Technique ◽  
Accretion Process

AbstractProteins evolve through the modular rearrangement of elements known as domains. It is hypothesized that extant, multidomain proteins are the result of domain accretion, but there has been limited experimental validation of this idea. Here, we introduce a technique for genetic minimization by iterative size-exclusion and recombination (MISER) that comprehensively assays all possible deletions of a protein. Using MISER, we generated a deletion landscape for the CRISPR protein Cas9. We found that Cas9 can tolerate large single deletions to the REC2, REC3, HNH, and RuvC domains, while still functioning in vitro and in vivo, and that these deletions can be stacked together to engineer minimal, DNA-binding effector proteins. In total, our results demonstrate that extant proteins retain significant modularity from the accretion process and, as genetic size is a major limitation for viral delivery systems, establish a general technique to improve genome editing and gene therapy-based therapeutics.

scholarly journals A rapid and tunable method to temporally control Cas9 expression enables the identification of essential genes and the interrogation of functional gene interactions in vitro and in vivo.

2015 ◽  
Author(s):  
Serif Senturk ◽  
Nitin H Shirole ◽  
Dawid D. Nowak ◽  
Vincenzo Corbo ◽  
Alexander Vaughan ◽  
...  
Keyword(s):  
Gene Editing ◽  
Essential Genes ◽  
Gene Interactions ◽  
Guide Rna ◽  
Functional Interactions ◽  
Cell Specificity ◽  
Efficient Gene ◽  
Systematic Identification

The Cas9/CRISPR system is a powerful tool for studying gene function. Here we describe a method that allows temporal control of Cas9/CRISPER activity based on conditional CAS9 destabilization. We demonstrate that fusing an FKBP12-derived destabilizing domain to Cas9 enables conditional rapid and reversible Cas9 expression in vitro and efficient gene-editing in the presence of a guide RNA. Further, we show that this strategy can be easily adapted to co-express, from the same promoter, DD-Cas9 with any other gene of interest, without the latter being co-modulated. In particular, when co-expressed with inducible Cre-ERT2, our system enables parallel, independent manipulation of alleles targeted by Cas9 and traditional recombinase with single-cell specificity. We anticipate this platform will be used for the systematic identification of essential genes and the interrogation of genes functional interactions.

scholarly journals TALEN Mediated Gene Targeting for CF Gene Therapy

2018 ◽  
Author(s):  
Emily Xia ◽  
Yiqian Zhang ◽  
Huibi Cao ◽  
Jun Li ◽  
Rongqi Duan ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Gene Editing ◽  
Airway Epithelial Cells ◽  
In Vivo Studies ◽  
Lacz Reporter ◽  
Gene Correction ◽  
Transcription Activator ◽  
Stringent Requirement

Cystic Fibrosis (CF) is an inherited monogenic disorder, amenable to gene based therapies. Because CF lung disease is currently the major cause of mortality and morbidity, and lung airway is readily accessible to gene delivery, the major CF gene therapy effort at present is directed to the lung. Although airway epithelial cells are renewed slowly, permanent gene correction through gene editing or targeting in airway stem cells is needed to perpetuate the therapeutic effect. Transcription activator-like effector nuclease (TALEN) has been utilized widely for a variety of gene editing applications. The stringent requirement for nuclease binding target sites allows for gene editing with precision. In this study, we engineered helper-dependent adenoviral (HD-Ad) vectors to deliver a pair of TALENs together with donor DNA targeting the human AAVS1 locus. With homology arms of 4 kb in length, we demonstrated precise insertion of either a LacZ reporter gene or a human CFTR minigene into the target site. Using the LacZ reporter, we determined the efficiency of gene integration to be about 5%. In the CFTR vector transduced cells, we have detected both CFTR mRNA and protein expression by qPCR and Wetern analysis, respectively. We have also confirmed CFTR function correction by flurometric Image Plate Reader (FLIPR) and iodide efflux assays. Taking together, these findings suggest a new direction for future in vitro and in vivo studies in CF gene editing.

scholarly journals All-in-One Adeno-associated Virus Delivery and Genome Editing by Neisseria meningitidis Cas9 in vivo

2018 ◽  
Author(s):  
Raed Ibraheim ◽  
Chun-Qing Song ◽  
Aamir Mir ◽  
Nadia Amrani ◽  
Wen Xue ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Neisseria Meningitidis ◽  
Genome Editing ◽  
Viral Vectors ◽  
Genome Engineering ◽  
Degradation Pathway ◽  
Type I ◽  
Guide Rna ◽  
Hereditary Tyrosinemia

AbstractClustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) have recently opened a new avenue for gene therapy. Cas9 nuclease guided by a single-guide RNA (sgRNA) has been extensively used for genome editing. Currently, three Cas9 orthologs have been adapted for in vivo genome engineering applications: SpyCas9, SauCas9 and CjeCas9. However, additional in vivo editing platforms are needed, in part to enable a greater range of sequences to be accessed via viral vectors, especially those in which Cas9 and sgRNA are combined into a single vector genome. Here, we present an additional in vivo editing platform using Neisseria meningitidis Cas9 (NmeCas9). NmeCas9 is compact, edits with high accuracy, and possesses a distinct PAM, making it an excellent candidate for safe gene therapy applications. We find that NmeCas9 can be used to target the Pcsk9 and Hpd genes in mice. Using tail vein hydrodynamic-based delivery of NmeCas9 plasmid to target the Hpd gene, we successfully reprogrammed the tyrosine degradation pathway in Hereditary Tyrosinemia Type I mice. More importantly, we delivered NmeCas9 with its single-guide RNA in a single recombinant adeno-associated vector (rAAV) to target Pcsk9, resulting in lower cholesterol levels in mice. This all-in-one vector yielded >35% gene modification after two weeks of vector administration, with minimal off-target cleavage in vivo. Our findings indicate that NmeCas9 can facilitate future efforts to correct disease-causing mutations by expanding the targeting scope of RNA-guided nucleases.

scholarly journals Genome Editing Technologies: From Bench Side to Bedside

Acta Medica ◽  
2018 ◽  
Vol 49 (3) ◽  
pp. 30
Author(s):  
Fulya Yaylacıoğlu Tuncay ◽  
Pervin Rukiye Dinçer
Keyword(s):  
Gene Therapy ◽  
Animal Models ◽  
Genome Editing ◽  
Clinical Studies ◽  
Human Gene ◽  
Human Gene Therapy ◽  
Novel Technologies ◽  
Preclinical And Clinical Studies

The development of genome editing technologies has given the chance to researchers to manipulate any genomic sequences precisely. This ability is very useful for creating animal models to study human diseases in vivo; for easy creation of isogenic cell lines to study in vitro and most importantly for overcoming many disadvantages that the researchers faced during the human gene therapy trials. Here we review the basic mechanisms of genome editing technology and the four genome-editing platforms. We also discuss the applications of these novel technologies in preclinical and clinical studies in four groups according to the mechanism used, and lastly, summarize the problems in these technologies.

scholarly journals CRISPR/Cpf1 mediated genome editing enhances Bombyx mori resistance to BmNPV

2020 ◽  
Author(s):  
Zhanqi Dong ◽  
Qi Qin ◽  
Zhigang Hu ◽  
Xinling Zhang ◽  
Jianghao Miao ◽  
...  
Keyword(s):  
Antiviral Therapy ◽  
Genome Editing ◽  
Genome Engineering ◽  
Transgenic Silkworm ◽  
Editing Efficiency ◽  
Transgenic Lines ◽  
Transgenic Silkworms ◽  
Bmnpv Genome

AbstractCRISPR/Cas12a (Cpf1) is a single RNA-guided endonuclease that provides new opportunities for targeted genome engineering through the CRISPR/Cas9 system. Only AsCpf1 have been developed for insect genome editing, and the novel Cas12a orthologs nucleases and editing efficiency require more study in insect. We compared three Cas12a orthologs nucleases, AsCpf1, FnCpf1, and LbCpf1, for their editing efficiencies and antiviral abilities in vitro. The three Cpf1 efficiently edited the BmNPV genome and inhibited BmNPV replication in BmN-SWU1 cells. The antiviral ability of the FnCpf1 system was more efficient than the SpCas9 system after infection by BmNPV. We created FnCpf1×gIE1 and SpCas9×sgIE1 transgenic hybrid lines and evaluated the gene editing efficiency of different systems at the same target site. We improved the antiviral ability using the FnCpf1 system in transgenic silkworm. This study demonstrated use of the CRISPR/Cpf1 system to achieve high editing efficiencies in the silkworm, and illustrates the use of this technology for increasing disease resistance.Author SummaryGenome editing is a powerful tool that has been widely used in gene function, gene therapy, pest control, and disease-resistant engineering in most parts of pathogens research. Since the establishment of CRISPR/Cas9, powerful strategies for antiviral therapy of transgenic silkworm have emerged. Nevertheless, there is still room to expand the scope of genome editing tool for further application to improve antiviral research. Here, we demonstrate that three Cpf1 endonuclease can be used efficiency editing BmNPV genome in vitro and in vivo for the first time. More importantly, this Cpf1 system could improve the resistance of transgenic silkworms to BmNPV compare with Cas9 system, and no significant cocoons difference was observed between transgenic lines infected with BmNPV and control. These broaden the range of application of CRISPR for novel genome editing methods in silkworm and also enable sheds light on antiviral therapy.

scholarly journals Plant viral vectors: Expanding the Possibilities of Precise Gene Editing in Plant Genomes

2021 ◽  
Author(s):  
Stuti Kujur ◽  
Muthappa Senthil-Kumar ◽  
Rahul Kumar
Keyword(s):  
Genome Editing ◽  
Plant Transformation ◽  
Viral Vectors ◽  
Gene Editing ◽  
Genome Engineering ◽  
Plant Cells ◽  
In Planta ◽  
Plant Genomes ◽  
Efficient Gene ◽  
Transformation Methods

Abstract The lack of a highly efficient method for delivering reagents for genome engineering to plant cells remains a bottleneck in achieving efficient gene-editing in plant genomes. A suite of recent reports uncovers the newly emerged roles of viral vectors, which can introduce gene-edits in plants with high mutation frequencies through in planta delivery. Here, we focus on the emerging protocols that utilized different approaches for virus-mediated genome editing in model plants. Testing of these protocols and the newly identified hypercompact Casɸ systems is needed to broaden the scope of genome-editing in most plant species, including crops, with minimized reliance on conventional plant transformation methods in the future.

Gene therapy development and legislation

2021 ◽  
Author(s):  
Moataz Dowaidar
Keyword(s):  
Gene Therapy ◽  
Genome Editing ◽  
Public Perception ◽  
Gene Editing ◽  
Ethical Issues ◽  
The Public ◽  
Ethical Violations ◽  
National Debate ◽  
Somatic Gene

As gene therapy and gene editing technologies improve, greater focus should be placed on techniques and procedures for treating heritable illnesses while respecting ethical norms. We're currently in the second cycle of public perception of gene therapy. During the 1990s gene therapy boom, the first surge of enthusiasm came to an end when the first gene therapy death occurred in 1999. While there are parallels between then and today in the public and scientific zeitgeist, the recall of earlier blunders has poisoned this assessment. Some are more recent, such as the 2018 announcement of germline-edited offspring, and stir up many more ethical arguments than this study can investigate.Despite academic disputes about the future of heritable genome editing, we are reaching a golden age of somatic gene therapy, with over 1,600 research presently active or recruiting patients. However, for both somatic and germline-based therapies, one thing appears to be universal: the scientific community must include the public and seek some type of broad societal discussion or consensus on widespread clinical use of gene editing. Governments should move promptly to provide public education on the scientific and ethical elements of genetic problems, allowing for national debate and universal consensus before legislation is enacted. These laws should seek to clarify ethical issues and reduce difficulties that may arise from somatic or germline genome editing, such as regulating the pricing of such medicines to make them affordable to all. After reviewing existing HHGE legislation worldwide, something like general societal agreement is already building, with 72 percent of nations exploring prohibiting HHGE. Currently, no country has legally authorized genome editing in vivo. Of course, this could change, especially as many nations begin to engage in a deeper dialog about HHGE with experts and the general public.What happens next will decide whether we can pursue this life-changing science responsibly or whether the dark matter of gene therapy, including ethical violations, will continue to plague the industry, leading to fresh cycles of hope, failure, and moratoriums.

scholarly journals Integrating Biomaterials and Genome Editing Approaches to Advance Biomedical Science

2021 ◽  
Vol 23 (1) ◽  
Author(s):  
Amr A. Abdeen ◽  
Brian D. Cosgrove ◽  
Charles A. Gersbach ◽  
Krishanu Saha
Keyword(s):  
Genome Editing ◽  
Gene Editing ◽  
Genome Engineering ◽  
Subsequent Development ◽  
Biomedical Science ◽  
Annual Review ◽  
Publication Date ◽  
Nonviral Delivery ◽  
Short Palindromic Repeat

The recent discovery and subsequent development of the clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated (Cas) platform as a precise genome editing tool have transformed biomedicine. As these CRISPR-based tools have matured, multiple stages of the gene editing process and the bioengineering of human cells and tissues have advanced. Here, we highlight recent intersections in the development of biomaterials and genome editing technologies. These intersections include the delivery of macromolecules, where biomaterial platforms have been harnessed to enable nonviral delivery of genome engineering tools to cells and tissues in vivo. Further, engineering native-like biomaterial platforms for cell culture facilitates complex modeling of human development and disease when combined with genome engineering tools. Deeper integration of biomaterial platforms in these fields could play a significant role in enabling new breakthroughs in the application of gene editing for the treatment of human disease. Expected final online publication date for the Annual Review of Biomedical Engineering, Volume 23 is June 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

scholarly journals Extensive adaptive immune response of AAVs and Cas proteins in non-human primates

2019 ◽  
Author(s):  
Puhao Xiao ◽  
Raoxian Bai ◽  
Ting Zhang ◽  
Yin Zhou ◽  
Zhigang Zhou ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Immune Response ◽  
Immune Responses ◽  
Gene Editing ◽  
Adaptive Immune Response ◽  
Gene Correction ◽  
Adaptive Immune ◽  
Correction System ◽  
Cas Proteins

AbstractThe CRISPR-mediated Cas system is the most widely used tool in gene editing and gene therapy for its convenience and efficiency. Delivery of the CRISPR system by adeno-associated viruses (AAVs) is currently the most promising approach to gene therapy. However, pre-existing adaptive immune responses against CRISPR nuclease (PAIR-C) and AAVs has been found in human serum, indicating that immune response is a problem that cannot be ignored, especially for in vivo gene correction. Non-human primates (NHPs) share many genetic and physiological traits with human, and are considered as the bridge for translational medicine. However, whether NHPs have same PAIR-C status with human is still unknown. Here, macaques (rhesus and cynomolgus), including normal housed and CRISPR-SpCas9 or TALENs edited individuals, were used to detect PAIR-C which covered SaCas9, SpCas9, AsCas12a and LbCas12a. Dogs and mice were also detected to expand the range of species. In addition, pre-existing adaptive antibodies to AAV8 and AAV9 were performed against macaques of different ages. The results showed that adaptive immunity was pre-existing in the macaques regardless of Cas proteins and AAVs. These findings indicate that the pre-existing adaptive immune of AAV-delivered CRISPR construction and correction system should be concerned for in vivo experiments.

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