scholarly journals In vivo genome editing in animals using AAV-CRISPR system: applications to translational research of human disease

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 2153 ◽  
Author(s):  
Cia-Hin Lau ◽  
Yousin Suh
Keyword(s):  
Clinical Trials ◽  
Genome Editing ◽  
Viral Vectors ◽  
Ex Vivo ◽  
Guide Rna ◽  
Routes Of Administration ◽  
Cas9 Nuclease ◽  
Wide Range ◽  
Short Palindromic Repeat

Adeno-associated virus (AAV) has shown promising therapeutic efficacy with a good safety profile in a wide range of animal models and human clinical trials. With the advent of clustered regulatory interspaced short palindromic repeat (CRISPR)-based genome-editing technologies, AAV provides one of the most suitable viral vectors to package, deliver, and express CRISPR components for targeted gene editing. Recent discoveries of smaller Cas9 orthologues have enabled the packaging of Cas9 nuclease and its chimeric guide RNA into a single AAV delivery vehicle for robust in vivo genome editing. Here, we discuss how the combined use of small Cas9 orthologues, tissue-specific minimal promoters, AAV serotypes, and different routes of administration has advanced the development of efficient and precise in vivo genome editing and comprehensively review the various AAV-CRISPR systems that have been effectively used in animals. We then discuss the clinical implications and potential strategies to overcome off-target effects, immunogenicity, and toxicity associated with CRISPR components and AAV delivery vehicles. Finally, we discuss ongoing non-viral-based ex vivo gene therapy clinical trials to underscore the current challenges and future prospects of CRISPR/Cas9 delivery for human therapeutics.

scholarly journals Nucleosomes inhibit target cleavage by CRISPR-Cas9 in vivo

2018 ◽  
Vol 115 (38) ◽  
pp. 9351-9358 ◽  
Author(s):  
Robert M. Yarrington ◽  
Surbhi Verma ◽  
Shaina Schwartz ◽  
Jonathan K. Trautman ◽  
Dana Carroll
Keyword(s):  
Genome Editing ◽  
Zinc Finger Nucleases ◽  
Yeast Genome ◽  
Target Sequence ◽  
Practical Applications ◽  
Guide Rna ◽  
Cas9 Protein ◽  
Wide Range

Genome editing with CRISPR-Cas nucleases has been applied successfully to a wide range of cells and organisms. There is, however, considerable variation in the efficiency of cleavage and outcomes at different genomic targets, even within the same cell type. Some of this variability is likely due to the inherent quality of the interaction between the guide RNA and the target sequence, but some may also reflect the relative accessibility of the target. We investigated the influence of chromatin structure, particularly the presence or absence of nucleosomes, on cleavage by the Streptococcus pyogenes Cas9 protein. At multiple target sequences in two promoters in the yeast genome, we find that Cas9 cleavage is strongly inhibited when the DNA target is within a nucleosome. This inhibition is relieved when nucleosomes are depleted. Remarkably, the same is not true of zinc-finger nucleases (ZFNs), which cleave equally well at nucleosome-occupied and nucleosome-depleted sites. These results have implications for the choice of specific targets for genome editing, both in research and in clinical and other practical applications.

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 In vivo CRISPR-Cas gene editing with no detectable genome-wide off-target mutations

2018 ◽  
Author(s):  
Pinar Akcakaya ◽  
Maggie L. Bobbin ◽  
Jimmy A. Guo ◽  
Jose M. Lopez ◽  
M. Kendell Clement ◽  
...  
Keyword(s):  
Genome Editing ◽  
Ex Vivo ◽  
Strong Support ◽  
Guide Rna ◽  
Genome Wide ◽  
Highly Sensitive ◽  
Further Development ◽  
Target Effects ◽  
Cas Gene

CRISPR-Cas genome-editing nucleases hold substantial promise for human therapeutics1–5 but identifying unwanted off-target mutations remains an important requirement for clinical translation6, 7. For ex vivo therapeutic applications, previously published cell-based genome-wide methods provide potentially useful strategies to identify and quantify these off-target mutation sites8–12. However, a well-validated method that can reliably identify off-targets in vivo has not been described to date, leaving the question of whether and how frequently these types of mutations occur. Here we describe Verification of In Vivo Off-targets (VIVO), a highly sensitive, unbiased, and generalizable strategy that we show can robustly identify genome-wide CRISPR-Cas nuclease off-target effects in vivo. To our knowledge, these studies provide the first demonstration that CRISPR-Cas nucleases can induce substantial off-target mutations in vivo, a result we obtained using a deliberately promiscuous guide RNA (gRNA). More importantly, we used VIVO to show that appropriately designed gRNAs can direct efficient in vivo editing without inducing detectable off-target mutations. Our findings provide strong support for and should encourage further development of in vivo genome editing therapeutic strategies.

scholarly journals Gene editing and CRISPR in the clinic: current and future perspectives

2020 ◽  
Vol 40 (4) ◽  
Author(s):  
Matthew P. Hirakawa ◽  
Raga Krishnakumar ◽  
Jerilyn A. Timlin ◽  
James P. Carney ◽  
Kimberly S. Butler
Keyword(s):  
Clinical Trials ◽  
Genome Editing ◽  
Dna Sequences ◽  
Gene Editing ◽  
Ex Vivo ◽  
Scientific Basis ◽  
Current Status ◽  
Zinc Finger Nucleases ◽  
Transcription Activator

Abstract Genome editing technologies, particularly those based on zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR (clustered regularly interspaced short palindromic repeat DNA sequences)/Cas9 are rapidly progressing into clinical trials. Most clinical use of CRISPR to date has focused on ex vivo gene editing of cells followed by their re-introduction back into the patient. The ex vivo editing approach is highly effective for many disease states, including cancers and sickle cell disease, but ideally genome editing would also be applied to diseases which require cell modification in vivo. However, in vivo use of CRISPR technologies can be confounded by problems such as off-target editing, inefficient or off-target delivery, and stimulation of counterproductive immune responses. Current research addressing these issues may provide new opportunities for use of CRISPR in the clinical space. In this review, we examine the current status and scientific basis of clinical trials featuring ZFNs, TALENs, and CRISPR-based genome editing, the known limitations of CRISPR use in humans, and the rapidly developing CRISPR engineering space that should lay the groundwork for further translation to clinical application.

scholarly journals Retron Editing for Precise Genome Editing without Exogenous Donor DNA in Human Cells

2021 ◽  
Author(s):  
Xiangfeng Kong ◽  
Zikang Wang ◽  
Yingsi Zhou ◽  
Xing Wang ◽  
Linyu Shi ◽  
...  
Keyword(s):  
Genome Editing ◽  
Ex Vivo ◽  
Human Cells ◽  
Exogenous Dna ◽  
Guide Rna ◽  
Homology Directed Repair ◽  
Non Coding Rna ◽  
Bacterial Genetic ◽  
Low Efficiency

CRISPR-Cas9 mediated seamless genome editing can be achieved by incorporating donor DNA into the CRISPR-Cas9 target loci via homology-directed repair (HDR), albeit with relative low efficiency due to the inefficient delivery of exogenous DNA. Retrons are bacterial genetic element composed of a non-coding RNA (ncRNA) and reverse transcriptase (RT). Retrons coupled with CRISPR-Cas9 have been shown to enhance precise genome editing via HDR in yeast through fusing guide RNA (gRNA) to the 3′ end of retron ncRNA, producing multicopy single-stranded DNA (msDNA) covalently tethered to gRNA. Here, we further engineered retrons by fusing Cas9 with E.coli RT from different clades and joining gRNA at the 5′ end of retron ncRNA, and found that retron editing can achieve precise genome editing efficiently in human cells. By co- expression of Cas9-RT fusions and retron-ncRNA gRNA (rgRNA) in HEK293T cells, we demonstrated the rates of retron editing at endogenous genomic loci was up to 10 %. We expect our retron editing system could aid in advancing the ex vivo and in vivo therapeutic applications of retron.

scholarly journals Preclinical and clinical advances in transposon-based gene therapy

2017 ◽  
Vol 37 (6) ◽  
Author(s):  
Jaitip Tipanee ◽  
Yoke Chin Chai ◽  
Thierry VandenDriessche ◽  
Marinee K. Chuah
Keyword(s):  
Gene Therapy ◽  
Clinical Trials ◽  
Viral Vectors ◽  
Ex Vivo ◽  
Cell Types ◽  
Sleeping Beauty ◽  
Target Cells ◽  
Hematologic Cancer ◽  
Clinical Advances

Transposons derived from Sleeping Beauty (SB), piggyBac (PB), or Tol2 typically require cotransfection of transposon DNA with a transposase either as an expression plasmid or mRNA. Consequently, this results in genomic integration of the potentially therapeutic gene into chromosomes of the desired target cells, and thus conferring stable expression. Non-viral transfection methods are typically preferred to deliver the transposon components into the target cells. However, these methods do not match the efficacy typically attained with viral vectors and are sometimes associated with cellular toxicity evoked by the DNA itself. In recent years, the overall transposition efficacy has gradually increased by codon optimization of the transposase, generation of hyperactive transposases, and/or introduction of specific mutations in the transposon terminal repeats. Their versatility enabled the stable genetic engineering in many different primary cell types, including stem/progenitor cells and differentiated cell types. This prompted numerous preclinical proof-of-concept studies in disease models that demonstrated the potential of DNA transposons for ex vivo and in vivo gene therapy. One of the merits of transposon systems relates to their ability to deliver relatively large therapeutic transgenes that cannot readily be accommodated in viral vectors such as full-length dystrophin cDNA. These emerging insights paved the way toward the first transposon-based phase I/II clinical trials to treat hematologic cancer and other diseases. Though encouraging results were obtained, controlled pivotal clinical trials are needed to corroborate the efficacy and safety of transposon-based therapies.

scholarly journals Therapeutic applications of CRISPR/Cas9 in breast cancer and delivery potential of gold nanomaterials

2020 ◽  
Vol 7 ◽  
pp. 184954352098319
Author(s):  
Jananee Padayachee ◽  
Moganavelli Singh
Keyword(s):  
Breast Cancer ◽  
Genome Editing ◽  
Target Cells ◽  
Guide Rna ◽  
Therapeutic Applications ◽  
Cas9 Nuclease ◽  
Current Therapies ◽  
Novel Therapeutic Strategies ◽  
Gold Nanomaterials

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.

Genome editing with CRISPR-Cas9: A budding biological contrivance for colorectal carcinoma research and its perspective in molecular medicine

2020 ◽  
Vol 20 ◽  
Author(s):  
Suman Kumar Ray ◽  
Sukhes Mukherjee
Keyword(s):  
Colon Cancer ◽  
Colorectal Carcinoma ◽  
Genome Editing ◽  
Gene Editing ◽  
Ex Vivo ◽  
Resistance Mechanism ◽  
Molecular Medicine ◽  
Guide Rna ◽  
Guide Rnas

: Genome editing is an addition, deletion, or replacement of a gene for wiping out or initiating explicit and preferred characters in the genome. Utilizing gene editing tools like CRISPR-Cas9 technology could be accomplished either by gene-based methodology or protein based technology that has been under scrutiny for protracted time wherein physical techniques, viral and non-viral strategies have been utilized together. Transplanting ex vivo CRISPR edited cells empowers screening of single guide RNAs with high-throughput and CRISPR based screening in organoids transplantation to validate cancer cells including colorectal carcinoma in various phases of its development and treatment. CRISPR knockout screens have recognized genes driving an interest in the colon cancer develop hallmarks, outstandingly for the disclosure of drug resistance mechanism in some cancer cell lines with single guide RNA. A benefit of this approach is to deal with genomic screening of CRISPR knockout, disrupts gene expression, rather than the partial knockdown which are frequently accomplished with RNA interference and CRISPR-Cas technology. Due to its proficient editing of the target gene, along with CRISPR/Cas system, this technique is used in the treatment of diverse types of cancer. In recent time research showed that CRISPR/Cas gene editing tool potentially reformed expression of long non-coding RNA in colorectal carcinoma. CRISPR/Cas9 technology will positively fuel the advancement of further in vivo gene editing clinical trials in colon cancer for forthcoming days and will have an immense impact in molecular medicine.

scholarly journals IMPROVING THE FUNCTION OF CRISPR-CAS9 FOR GENOME EDITING THERAPY: EDITING THE EDITOR

2017 ◽  
Vol 4 (1) ◽  
pp. 44
Author(s):  
Alva Sahiri Alexander Supit
Keyword(s):  
Genome Editing ◽  
Viral Vectors ◽  
Gene Editing ◽  
Genetic Disorders ◽  
Genetic Diseases ◽  
End Joining ◽  
Guide Rna ◽  
Homology Directed Repair ◽  
Short Palindromic Repeat ◽  
Active Research

Meningkatkan Fungsi CRISPR-Cas9 untuk Terapi Pengeditan GenPengeditan gen menjadi mudah dilakukan sejak ditemukannya clustered regularly interspaced short palindromic repeat (CRISPR) dan CRISPR-associated protein 9 (Cas9) sebagai alat untuk menyunting gen suatu organisme. Sebagian besar penyakit genetik tidak dapat disembuhkan secara kausal dengan terapi yang ada, maka pengeditan gen merupakan suatu cara yang prospektif dalam terapi medis di masa depan. Sayangnya, pengeditan gen dengan Cas9 yang ada saat ini masih memiliki banyak kelemahan, yaitu: 1) kurang spesifik, di mana RNA pemandu dapat berikatan dengan beberapa segmen pada genom manusia, sehingga memungkinkan terjadinya salah target; 2) kurang efisien, karena sekalipun telah berhasil memotong utas ganda DNA, kebanyakan penyambungan kembali akan dilakukan secara non-homology end joining (NHEJ), yang justru meningkatkan peluang terjadinya mutasi; 3) sulit disalurkan ke dalam inti sel karena berbagai sawar fisiologis maupun biokimiawi. Tulisan ini akan membahas perkembangan terkini dalam mengatasi ketiga masalah di atas. Untuk meningkatkan spesifisitas, dapat dilakukan modifikasi RNA pemandu dan struktur Cas9. Efisiensi dapat ditingkatkan dengan meningkatkan peluang terjadinya homology-directed repair dibandingkan NHEJ, sedangkan untuk meningkatkan distribusi ke dalam sel, dapat digunakan berbagai macam vektor, seperti virus dan nanopartikel. CRISPR-Cas9 merupakan area yang aktif diteliti dalam bidang biosains, dan dalam waktu dekat, diharapkan dapat dimanfaatkan dalam bidang klinik.Kata kunci: CRISPR, Cas9, efektivitas, spesifisitas, terapi genABSTRACTGene editing has become reasonably easy since the discovery of clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9). Most genetic diseases cannot be treated causally, and currently available therapies are mainly symptom-based. To treat the etiology of genetic diseases, a firm gene editing therapy is necessary to be established. This posits Cas9-facilitated gene editing as a prospective modality to become a clinically approved therapy in the future to treat genetic disorders. However, until recently, Cas9-based genome editing is still facing several hurdles, including low specificity, low effectiveness, and difficult delivery. Currently available Cas9 nucleases are able to bind to non-specific DNA sequence and produce non-specific cleavage. The efficiency has been relatively low due to the preference of non-homologous end-joining (NHEJ) over homology-directed repair (HDR) by the host cell. Furthermore, in order to deliver Cas9 into the nucleus, multiple physiological barriers have to be overcome. This review discussed recent developments in tackling these three hurdles, ranging from designing the guide RNA using multiple bioinformatics tools, modifying Cas9 structure, as well as packaging the nuclease-guide RNA complex into viral vectors and nanoparticles. Considering the active research on this area, it is expected that CRISPR/Cas9 can be utilized as a clinical therapy in the near future.Received: 02 June 2017        Accepted: 07 July 2017        Published: 19 July 2017

Development and Challenges of Nanovectors in Gene Therapy

Nano LIFE ◽  
2014 ◽  
Vol 04 (03) ◽  
pp. 1441007 ◽  
Author(s):  
Yarong Liu ◽  
Jennifer Rohrs ◽  
Pin Wang
Keyword(s):  
Gene Therapy ◽  
Clinical Trials ◽  
Gene Delivery ◽  
Viral Vectors ◽  
Nonviral Vectors ◽  
Cellular Compartment ◽  
Current State ◽  
Gene Delivery Vectors ◽  
Wide Range

In recent years, hundreds of genes have been linked to a variety of human diseases, and the field of gene therapy has emerged as a way to treat this wide range of diseases. The main goal of gene therapy is to find a gene delivery vehicle that can successfully target diseased cells and deliver therapeutic genes directly to their cellular compartment. The two main types of gene delivery vectors currently being investigated in clinical trials are recombinant viral vectors and synthetic nonviral vectors. Recombinant viral vectors take advantage of the evolutionarily optimized viral mechanisms to deliver genes, but they can be hard to specifically target in vivo and are also associated with serious side effects. Synthetic nonviral vectors are made out of highly biocompatible lipids or polymers, but they are much less efficient at delivering their genetic payload due to the lack of any active delivery mechanism. This mini review will introduce the current state of gene delivery in clinical trials, and discuss the specific challenges associated with each of these vectors. It will also highlight some specific gaps in knowledge that are limiting the advancement of this field and touch on the current areas of research being explored to overcome them.

Sign in / Sign up

Export Citation Format

Share Document