scholarly journals Applications and challenges of CRISPR-Cas gene-editing to disease treatment in clinics

2021 ◽  
Author(s):  
Wenyi Liu ◽  
Luoxi Li ◽  
Jianxin Jiang ◽  
Min Wu ◽  
Ping Lin
Keyword(s):  
Gene Editing ◽  
Hereditary Diseases ◽  
Laboratory Research ◽  
Great Promise ◽  
Clinical Tests ◽  
Disease Treatment ◽  
Relative Difficulty ◽  
Agricultural Engineering ◽  
Target Effects ◽  
Cas Gene

Abstract Clustered regularly interspaced short palindromic repeats-CRISPR associated systems (Cas) are efficient tools for targeting specific genes for laboratory research, agricultural engineering, biotechnology, and human disease treatment. Cas9, by far the most extensively used gene-editing nuclease, has shown great promise for the treatment of hereditary diseases, viral infection, cancers and so on. Recent reports have revealed that some other types of CRISPR-Cas systems may also have surprising potential to join the fray as gene-editing tools for various applications. Despite the fast progress in basic researches and clinical tests, some underlying problems present continuous, significant challenges, such as editing efficiency, relative difficulty in delivery, off-target effects, immunogenicity, etc. This article summarizes the applications of CRISPR-Cas from bench to bedside and highlights the current obstacles that may limit the usage of CRISPR-Cas systems as gene-editing tool-kits in precision medicine and offer some viewpoints that may help to tackle these challenges and facilitate technical development. CRISPR-Cas systems, as a powerful gene-editing approach, will offer great hopes in clinical treatments for many individuals with currently incurable diseases.

scholarly journals Nanoparticle Delivery of CRISPR/Cas9 for Genome Editing

2021 ◽  
Vol 12 ◽  
Author(s):  
Li Duan ◽  
Kan Ouyang ◽  
Xiao Xu ◽  
Limei Xu ◽  
Caining Wen ◽  
...  
Keyword(s):  
System Development ◽  
Delivery Systems ◽  
Inorganic Nanoparticles ◽  
Great Promise ◽  
The Novel ◽  
Disease Treatment ◽  
Nanoparticle Delivery ◽  
Promising Tool ◽  
Short Palindromic Repeat ◽  
Cas Gene

The emerging clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated system (Cas) gene-editing system represents a promising tool for genome manipulation. However, its low intracellular delivery efficiency severely compromises its use and potency for clinical applications. Nanocarriers, such as liposomes, polymers, and inorganic nanoparticles, have shown great potential for gene delivery. The remarkable development of nanoparticles as non-viral carriers for the delivery of the CRISPR/Cas9 system has shown great promise for therapeutic applications. In this review, we briefly summarize the delivery components of the CRISPR/Cas9 system and report on the progress of nano-system development for CRISPR/Cas9 delivery. We also compare the advantages of various nano-delivery systems and their applications to deliver CRISPR/Cas9 for disease treatment. Nano-delivery systems can be modified to fulfill the tasks of targeting cells or tissues. We primarily emphasize the novel exosome-based CRISPR/Cas9 delivery system. Overall, we review the challenges, development trends, and application prospects of nanoparticle-based technology for CRISPR/Cas9 delivery.

Clustered regularly interspaced short palindromic repeats, a glimpse – impacts in molecular biology, trends and highlights

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Dhivya Selvaraj ◽  
Rajni Dawar ◽  
Pradeep Kumar Sivakumar ◽  
Anita Devi
Keyword(s):  
Molecular Biology ◽  
Infectious Diseases ◽  
Gene Editing ◽  
Inflammatory Diseases ◽  
Genetic Diseases ◽  
Hereditary Diseases ◽  
Hemorrhagic Fevers ◽  
Viral Hemorrhagic Fevers ◽  
Target Effects ◽  
Working Mechanisms

Abstract Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is a novel molecular tool. In recent days, it has been highlighted a lot, as the Nobel prize was awarded for this sector in 2020, and also for its recent use in Covid-19 related diagnostics. Otherwise, it is an eminent gene-editing technique applied in diverse medical zones of therapeutics in genetic diseases, hematological diseases, infectious diseases, etc., research related to molecular biology, cancer, hereditary diseases, immune and inflammatory diseases, etc., diagnostics related to infectious diseases like viral hemorrhagic fevers, Covid-19, etc. In this review, its discovery, working mechanisms, challenges while handling the technique, recent advancements, applications, alternatives have been discussed. It is a cheaper, faster technique revolutionizing the medicinal field right now. However, their off-target effects and difficulties in delivery into the desired cells make CRISPR, not easily utilizable. We conclude that further robust research in this field may promise many interesting, useful results.

CRISPR Systems Suitable for Single AAV Vector Delivery

2021 ◽  
Vol 21 ◽  
Author(s):  
Marta Stevanovic ◽  
Elena Piotter ◽  
Michelle McClements ◽  
Robert MacLaren
Keyword(s):  
Gene Editing ◽  
Great Promise ◽  
Genetic Mutations ◽  
Aav Vector ◽  
Guide Rna ◽  
Packaging Capacity ◽  
Vector Delivery ◽  
Cas Gene ◽  
Cas Proteins

: CRISPR (clustered regularly interspaced short palindromic repeats)/Cas gene editing is a revolutionary technology that can enable the correction of genetic mutations in vivo, providing great promise as a therapeutic intervention for inherited diseases. Adeno-associated viral (AAV) vectors are a potential vehicle for delivering CRISPR/Cas. However, they are restricted by their limited packaging capacity. Identifying smaller Cas orthologs that can be packaged, along with the required guide RNA elements, into a single AAV would be an important optimization for CRISPR/Cas gene editing. Expanding the options of Cas proteins that can be delivered by a single AAV not only increases translational application but also expands the genetic sites that can be targeted for editing. This review considers the benefits and current scope of small Cas protein orthologs that are suitable for gene editing approaches using single AAV vector delivery.

scholarly journals Spatiotemporal control of CRISPR/Cas9 gene editing

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Chenya Zhuo ◽  
Jiabin Zhang ◽  
Jung-Hwan Lee ◽  
Ju Jiao ◽  
Du Cheng ◽  
...  
Keyword(s):  
Genetic Engineering ◽  
Gene Editing ◽  
Genetic Regulation ◽  
Clinical Translation ◽  
Small Molecule Activation ◽  
Simple Design ◽  
Advantages And Disadvantages ◽  
Spatiotemporal Control ◽  
Critical Challenge ◽  
Target Effects

AbstractThe clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (CRISPR/Cas9) gene editing technology, as a revolutionary breakthrough in genetic engineering, offers a promising platform to improve the treatment of various genetic and infectious diseases because of its simple design and powerful ability to edit different loci simultaneously. However, failure to conduct precise gene editing in specific tissues or cells within a certain time may result in undesirable consequences, such as serious off-target effects, representing a critical challenge for the clinical translation of the technology. Recently, some emerging strategies using genetic regulation, chemical and physical strategies to regulate the activity of CRISPR/Cas9 have shown promising results in the improvement of spatiotemporal controllability. Herein, in this review, we first summarize the latest progress of these advanced strategies involving cell-specific promoters, small-molecule activation and inhibition, bioresponsive delivery carriers, and optical/thermal/ultrasonic/magnetic activation. Next, we highlight the advantages and disadvantages of various strategies and discuss their obstacles and limitations in clinical translation. Finally, we propose viewpoints on directions that can be explored to further improve the spatiotemporal operability of CRISPR/Cas9.

scholarly journals Human Genetic Diversity Alters Therapeutic Gene Editing Off-Target Outcomes

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3993-3993
Author(s):  
Linda Yingqi Lin ◽  
Samuele Cancellieri ◽  
Jing Zeng ◽  
Francesco Masillo ◽  
My Anh Nguyen ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Clinical Trials ◽  
Genome Editing ◽  
Gene Editing ◽  
Conflicts Of Interest ◽  
African Ancestry ◽  
Great Promise ◽  
Personal Genome ◽  
Hematopoietic Stem ◽  
The Impact

Abstract CRISPR gene editing holds great promise to modify somatic genomes to ameliorate disease. In silico prediction of homologous sites coupled with biochemical evaluation of possible genomic off-targets may predict genotoxicity risk of individual gene editing reagents. However, standard computational and biochemical methods focus on reference genomes and do not consider the impact of genetic diversity on off-target potential. Here we developed a web application called CRISPRme that explicitly and efficiently integrates human genetic variant datasets with orthogonal genomic annotations to predict and prioritize off-target sites at scale. The method considers both single-nucleotide variants (SNVs) and indels, accounts for bona fide haplotypes, accepts spacer:protospacer mismatches and bulges, and is suitable for personal genome analyses. We tested the tool with a guide RNA (gRNA) targeting the BCL11A erythroid enhancer that has shown therapeutic promise in clinical trials for sickle cell disease (SCD) and β-thalassemia (Frangoul et al. NEJM 2021). We find that the top predicted off-target site is produced by a non-reference allele common in African-ancestry populations (rs114518452, minor allele frequency (MAF) = 4.5%) that introduces a protospacer adjacent motif (PAM) for SpCas9. We validate that SpCas9 generates indels (~9.6% frequency) and chr2 pericentric inversions in a strictly allele-specific manner in edited CD34+ hematopoietic stem/progenitor cells (HSPCs), although a high-fidelity Cas9 variant mitigates this off-target. This report illustrates how population and private genetic variants should be considered as modifiers of genome editing outcomes. We expect that variant-aware off-target assessment will be required for therapeutic genome editing efforts going forward, including both ongoing and future clinical trials, and we provide a powerful approach for comprehensive off-target prediction. CRISPRme is available at crisprme.di.univr.it. Disclosures No relevant conflicts of interest to declare.

CRISPR/Cas9 for cancer treatment: technology, clinical applications and challenges

2020 ◽  
Vol 19 (3) ◽  
pp. 209-214 ◽  
Author(s):  
Xing Cheng ◽  
Shaoyi Fan ◽  
Chengcai Wen ◽  
Xianfa Du
Keyword(s):  
Cancer Treatment ◽  
Viral Infections ◽  
Adaptive Immune System ◽  
Clinical Applications ◽  
Great Promise ◽  
General Function ◽  
Treatment Technology ◽  
Delivery Methods ◽  
Function Mechanism ◽  
Target Effects

Abstract Clustered regularly interspaced short palindromic repeats (CRISPR) is described as RNA mediated adaptive immune system defense, which is naturally found in bacteria and archaea. CRISPR-Cas9 has shown great promise for cancer treatment in cancer immunotherapy, manipulation of cancer genome and epigenome and elimination or inactivation of carcinogenic viral infections. However, many challenges remain to be addressed to increase its efficacy, including off-target effects, editing efficiency, fitness of edited cells, immune response and delivery methods. Here, we explain CRISPR-Cas classification and its general function mechanism for gene editing. Then, we summarize these preclinical CRISPR-Cas9-based therapeutic strategies against cancer. Moreover, the challenges and improvements of CRISPR-Cas9 clinical applications will be discussed.

scholarly journals The Diversity of Genetic Outcomes from CRISPR/Cas Gene Editing is Regulated by the Length of the Symmetrical Donor DNA Template

Genes ◽  
2020 ◽  
Vol 11 (10) ◽  
pp. 1160
Author(s):  
Amanda M. Hewes ◽  
Brett M. Sansbury ◽  
Eric B. Kmiec
Keyword(s):  
Gene Editing ◽  
Bacterial Cells ◽  
Knock Out ◽  
Viral Genomes ◽  
Homology Directed Repair ◽  
Established Cell ◽  
Dna Template ◽  
Donor Template ◽  
Eukaryotic Genomes ◽  
Cas Gene

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas gene editing systems have enabled molecular geneticists to manipulate prokaryotic and eukaryotic genomes with greater efficiency and precision. CRISPR/Cas provides adaptive immunity in bacterial cells by degrading invading viral genomes. By democratizing this activity into human cells, it is possible to knock out specific genes to disable their function and repair errors. The latter of these activities requires the participation of a single-stranded donor DNA template that provides the genetic information to execute correction in a process referred to as homology directed repair (HDR). Here, we utilized an established cell-free extract system to determine the influence that the donor DNA template length has on the diversity of products from CRISPR-directed gene editing. This model system enables us to view all outcomes of this reaction and reveals that donor template length can influence the efficiency of the reaction and the categories of error-prone products that accompany it. A careful measurement of the products revealed a category of error-prone events that contained the corrected template along with insertions and deletions (indels). Our data provides foundational information for those whose aim is to translate CRISPR/Cas from bench to bedside.

scholarly journals Proteases as therapeutics

2011 ◽  
Vol 435 (1) ◽  
pp. 1-16 ◽  
Author(s):  
Charles S. Craik ◽  
Michael J. Page ◽  
Edwin L. Madison
Keyword(s):  
Clinical Application ◽  
Molecular Mechanisms ◽  
New Technologies ◽  
Great Promise ◽  
Disease Treatment ◽  
Therapeutic Class ◽  
Potential Applications ◽  
Plasma Fractions ◽  
History Of ◽  
Delivery Options

Proteases are an expanding class of drugs that hold great promise. The U.S. FDA (Food and Drug Administration) has approved 12 protease therapies, and a number of next generation or completely new proteases are in clinical development. Although they are a well-recognized class of targets for inhibitors, proteases themselves have not typically been considered as a drug class despite their application in the clinic over the last several decades; initially as plasma fractions and later as purified products. Although the predominant use of proteases has been in treating cardiovascular disease, they are also emerging as useful agents in the treatment of sepsis, digestive disorders, inflammation, cystic fibrosis, retinal disorders, psoriasis and other diseases. In the present review, we outline the history of proteases as therapeutics, provide an overview of their current clinical application, and describe several approaches to improve and expand their clinical application. Undoubtedly, our ability to harness proteolysis for disease treatment will increase with our understanding of protease biology and the molecular mechanisms responsible. New technologies for rationally engineering proteases, as well as improved delivery options, will expand greatly the potential applications of these enzymes. The recognition that proteases are, in fact, an established class of safe and efficacious drugs will stimulate investigation of additional therapeutic applications for these enzymes. Proteases therefore have a bright future as a distinct therapeutic class with diverse clinical applications.

scholarly journals Optimized RNP transfection for highly efficient CRISPR/Cas9-mediated gene knockout in primary T cells

2018 ◽  
Vol 215 (3) ◽  
pp. 985-997 ◽  
Author(s):  
Akiko Seki ◽  
Sascha Rutz
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Gene Editing ◽  
Target Gene ◽  
Gene Knockout ◽  
Great Promise ◽  
Next Generation ◽  
Primary Mouse

CRISPR (clustered, regularly interspaced, short palindromic repeats)/Cas9 (CRISPR-associated protein 9) has become the tool of choice for generating gene knockouts across a variety of species. The ability for efficient gene editing in primary T cells not only represents a valuable research tool to study gene function but also holds great promise for T cell–based immunotherapies, such as next-generation chimeric antigen receptor (CAR) T cells. Previous attempts to apply CRIPSR/Cas9 for gene editing in primary T cells have resulted in highly variable knockout efficiency and required T cell receptor (TCR) stimulation, thus largely precluding the study of genes involved in T cell activation or differentiation. Here, we describe an optimized approach for Cas9/RNP transfection of primary mouse and human T cells without TCR stimulation that results in near complete loss of target gene expression at the population level, mitigating the need for selection. We believe that this method will greatly extend the feasibly of target gene discovery and validation in primary T cells and simplify the gene editing process for next-generation immunotherapies.

scholarly journals The technical risks of human gene editing

2019 ◽  
Vol 34 (11) ◽  
pp. 2104-2111 ◽  
Author(s):  
Benjamin Davies
Keyword(s):  
Genome Editing ◽  
Gene Editing ◽  
Human Gene ◽  
Ethical Concerns ◽  
Technical Risks ◽  
Genomic Mutation ◽  
Cas Gene

Abstract A recent report from Dr He Jiankui concerning the birth of twin girls harbouring mutations engineered by CRISPR/Cas nucleases has been met with international condemnation. Beside the serious ethical concerns, there are known technical risks associated with CRISPR/Cas gene editing which further raise questions about how these events could have been allowed to occur. Numerous studies have reported unexpected genomic mutation and mosaicism following the use of CRISPR/Cas nucleases, and it is currently unclear how prevalent these disadvantageous events are and how robust and sensitive the strategies to detect these unwanted events may be. Although Dr Jiankui’s study appears to have involved certain checks to ascertain these risks, the decision to implant the manipulated embryos, given these unknowns, must nonetheless be considered reckless. Here I review the technical concerns surrounding genome editing and consider the available data from Dr Jiankui in this context. Although the data remains unpublished, preventing a thorough assessment of what was performed, it seems clear that the rationale behind the undertaking was seriously flawed; the procedures involved substantial technical risks which, when added to the serious ethical concerns, fully justify the widespread criticism that the events have received.

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