Cas9 conjugate complex delivering donor DNA for efficient gene editing by homology-directed repair

2021 ◽  
Author(s):  
Yoo Kyung Kang ◽  
Ju Hee Lee ◽  
San Hae Im ◽  
Joo Hoon Lee ◽  
Juhee Jeong ◽  
...  
Keyword(s):  
Gene Editing ◽  
Homology Directed Repair ◽  
Efficient Gene

Enhanced Homology-directed Repair for Highly Efficient Gene Editing in Hematopoietic Stem/Progenitor Cells

Blood ◽  
2021 ◽  
Author(s):  
Suk See De Ravin ◽  
Julie Brault ◽  
Ronald J Meis ◽  
Siyuan Liu ◽  
Linhong Li ◽  
...  
Keyword(s):  
Gene Editing ◽  
Hematopoietic Stem ◽  
Vector Integration ◽  
Homology Directed Repair ◽  
Highly Efficient ◽  
Donor Control ◽  
Efficient Gene ◽  
Cd34 Cells ◽  
Viable Approach

Lentivector gene therapy for X-linked chronic granulomatous disease (X-CGD) has proven to be a viable approach, but random vector integration and subnormal protein production from exogenous promoters in transduced cells remain concerning for long-term safety and efficacy. A previous genome editing-based approach using SpCas9 and an oligodeoxynucleotide donor to repair genetic mutations demonstrated the capability to restore physiological protein expression, but lacked sufficient efficiency in quiescent CD34+ hematopoietic cells for clinical translation. Here, we show transient inhibition of p53-binding protein 1 (53BP1) significantly increased (2.3-fold) long-term homology directed repair (HDR) to achieve highly efficient (80% gp91phox+ cells compared to healthy donor control) long-term correction of X-CGD CD34+ cells.

scholarly journals Fast, efficient and virus-free generation of TRAC-replaced CAR T cells

2021 ◽  
Author(s):  
Jonas Kath ◽  
Weijie Du ◽  
Bernice Thommandru ◽  
Rolf Turk ◽  
Leila Amini ◽  
...  
Keyword(s):  
T Cells ◽  
Gene Editing ◽  
Cell Receptor ◽  
Homology Directed Repair ◽  
Car T Cells ◽  
Human T Cells ◽  
Efficient Gene ◽  
Car T ◽  
Specific Cytotoxicity

AbstractChimeric Antigen Receptor (CAR) redirected T cells are a potent treatment option for certain hematological malignancies. Recently, site-specific insertion of CARs into the T cell receptor (TCR) alpha constant (TRAC) locus using gene editing and adeno-associated viruses was shown to generate CAR T cells with improved functionality over their retrovirally transduced counterparts. However, the development of viruses for gene transfer is complex and associated with extensive costs at early clinical stages. Here, we provide an economical and virus-free method for efficient CAR insertion into the TRAC locus of primary human T cells via CRISPR-Cas mediated homology-directed repair (HDR). While the toxicity induced by transfected double-stranded template (donor) DNA was not fully prevented by pharmacological means, the combination of DNA-sensor inhibitors and HDR enhancers resulted in highly efficient gene editing with TCR-to-CAR replacement rates reaching up to 68%. The resulting TCR-deficient CAR T cells show antigen-specific cytotoxicity and cytokine production in vitro. Our GMP-compatible non-viral platform technology lays the foundation for clinical trials and fast-track generation of novel CAR T cells applicable for autologous or allogeneic off-the-shelf use.

scholarly journals Simplified All-In-One CRISPR-Cas9 Construction for Efficient Genome Editing in Cryptococcus Species

2021 ◽  
Vol 7 (7) ◽  
pp. 505
Author(s):  
Ping Zhang ◽  
Yu Wang ◽  
Chenxi Li ◽  
Xiaoyu Ma ◽  
Lan Ma ◽  
...  
Keyword(s):  
Genome Editing ◽  
Fungal Pathogens ◽  
Gene Editing ◽  
Recombination Frequency ◽  
Target Sequence ◽  
Widespread Application ◽  
Genetic Studies ◽  
Efficient Gene ◽  
Cryptococcus Species ◽  
Overlap Pcr

Cryptococcus neoformans and Cryptococcus deneoformans are opportunistic fungal pathogens found worldwide that are utilized to reveal mechanisms of fungal pathogenesis. However, their low homologous recombination frequency has greatly encumbered genetic studies. In preliminary work, we described a ‘suicide’ CRISPR-Cas9 system for use in the efficient gene editing of C. deneoformans, but this has not yet been used in the C. neoformans strain. The procedures involved in constructing vectors are time-consuming, whether they involve restriction enzyme-based cloning of donor DNA or the introduction of a target sequence into the gRNA expression cassette via overlap PCR, as are sophisticated, thus impeding their widespread application. Here, we report the optimized and simplified construction method for all-in-one CRISPR-Cas9 vectors that can be used in C. neoformans and C. deneoformans strains respectively, named pNK003 (Genbank: MW938321) and pRH003 (Genbank: KX977486). Taking several gene manipulations as examples, we also demonstrate the accuracy and efficiency of the new simplified all-in-one CRISPR-Cas9 genome editing tools in both Serotype A and Serotype D strains, as well as their ability to eliminate Cas9 and gDNA cassettes after gene editing. We anticipate that the availability of new vectors that can simplify and streamline the technical steps for all-in-one CRISPR-Cas9 construction could accelerate genetic studies of the Cryptococcus species.

scholarly journals Design of time-delayed safety switches for CRISPR gene therapy

2021 ◽  
Author(s):  
Dashan Sun
Keyword(s):  
Gene Therapy ◽  
Immune Response ◽  
Time Delay ◽  
Cell Lines ◽  
Gene Editing ◽  
Drug Accumulation ◽  
Work Time ◽  
Crispr System ◽  
Efficient Gene ◽  
Target Effect

CRISPR system is a powerful gene editing tool which has already been reported to address a variety of gene relevant diseases in different cell lines. However, off-target effect and immune response caused by Cas9 remain two fundamental problems. In our work, time-delayed safety switches are designed based on either artificial ultrasensitivity transmission module or intrinsic time delay in biomolecular activities. By addressing gene therapy efficiency, off-target effect, immune response and drug accumulation, we hope our safety switches may offer inspiration in realizing safe and efficient gene therapy in humans.

PML isoform expression and DNA break location relative to PML nuclear bodies impacts the efficiency of homologous recombination

2020 ◽  
Vol 98 (3) ◽  
pp. 314-326 ◽  
Author(s):  
Kathleen M. Attwood ◽  
Jayme Salsman ◽  
Dudley Chung ◽  
Sabateeshan Mathavarajah ◽  
Carter Van Iderstine ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Gene Editing ◽  
Genotoxic Stress ◽  
Nuclear Bodies ◽  
Homology Directed Repair ◽  
Pml Nuclear Bodies ◽  
Promyelocytic Leukemia Nuclear Bodies ◽  
Chromosomal Break ◽  
The Impact

Promyelocytic leukemia nuclear bodies (PML NBs) are nuclear subdomains that respond to genotoxic stress by increasing in number via changes in chromatin structure. However, the role of the PML protein and PML NBs in specific mechanisms of DNA repair has not been fully characterized. Here, we have directly examined the role of PML in homologous recombination (HR) using I-SceI extrachromosomal and chromosome-based homology-directed repair (HDR) assays, and in HDR by CRISPR/Cas9-mediated gene editing. We determined that PML loss can inhibit HR in an extrachromosomal HDR assay but had less of an effect on CRISPR/Cas9-mediated chromosomal HDR. Overexpression of PML also inhibited both CRISPR HDR and I-SceI-induced HDR using a chromosomal reporter, and in an isoform-specific manner. However, the impact of PML overexpression on the chromosomal HDR reporter was dependent on the intranuclear chromosomal positioning of the reporter. Specifically, HDR at the TAP1 gene locus, which is associated with PML NBs, was reduced compared with a locus not associated with a PML NB; yet, HDR could be reduced at the non-PML NB-associated locus by PML overexpression. Thus, both loss and overexpression of PML isoforms can inhibit HDR, and proximity of a chromosomal break to a PML NB can impact HDR efficiency.

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 Exploration of CRISPR/Cas9-based gene editing as therapy for osteoarthritis

2019 ◽  
Vol 78 (5) ◽  
pp. 676-682 ◽  
Author(s):  
Lan Zhao ◽  
Jian Huang ◽  
Yunshan Fan ◽  
Jun Li ◽  
Tianming You ◽  
...  
Keyword(s):  
Structural Damage ◽  
Drug Targets ◽  
Gene Editing ◽  
Joint Damage ◽  
Loss Of Function ◽  
Adeno Associated Virus ◽  
Structural Deterioration ◽  
Efficient Gene ◽  
Genes Encoding ◽  
Surgically Induced

ObjectivesOsteoarthritis (OA) is a painful and debilitating disease and it is associated with aberrant upregulation of multiple factors, including matrix metalloproteinase 13 (MMP13), interleukin-1β (IL-1β) and nerve growth factor (NGF). In this study, we aimed to use the CRISPR/Cas9 technology, a highly efficient gene-editing tool, to study whether the ablation of OA-associated genes has OA-modifying effects.MethodsWe performed intra-articular injection of adeno-associated virus, which expressed CRISPR/Cas9 components to target each of the genes encoding MMP13, IL-1β and NGF, in a surgically induced OA mouse model. We also tested triple ablations of NGF, MMP13 and IL-1β.ResultsLoss-of-function of NGF palliates pain but worsens joint damage in the surgically induced OA model. Ablation of MMP13 or IL-1β reduces the expression of cartilage-degrading enzymes and attenuates structural deterioration. Targeting both MMP13 and IL-1β significantly mitigates the adverse effects of NGF blockade on the joints.ConclusionsCRISPR-mediated ablation of NGF alleviates OA pain, and deletion of MMP13-1β or IL-1β attenuates structural damage in a post-traumatic OA model. Multiplex ablations of NGF, MMP13 and IL-1β provide benefits on both pain management and joint structure maintenance. Our results suggest that CRISPR-based gene editing is useful for the identification of promising drug targets and the development of feasible therapeutic strategies for OA treatment.

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