Efficient Gene Editing in Tomato in the First Generation Using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-Associated9 System

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.

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Design of time-delayed safety switches for CRISPR gene therapy

10.1101/2021.04.17.440285 ◽

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.

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

Journal of Industrial and Engineering Chemistry ◽

10.1016/j.jiec.2021.07.009 ◽

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

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714. Structure-Based Engineering of the FokI DNA Cleavage Domain in the Context of a Chimeric Endonuclease: Towards Maximally Efficient “Gene Editing” Therapy

Molecular Therapy ◽

10.1016/j.ymthe.2004.06.620 ◽

2004 ◽

Vol 9 ◽

pp. S272

Keyword(s):

Dna Cleavage ◽

Gene Editing ◽

Efficient Gene ◽

Cleavage Domain

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

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2018-214724 ◽

2019 ◽

Vol 78 (5) ◽

pp. 676-682 ◽

Cited By ~ 22

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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Sendai virus, an RNA virus with no risk of genomic integration, delivers CRISPR/Cas9 for efficient gene editing

Molecular Therapy — Methods & Clinical Development ◽

10.1038/mtm.2016.57 ◽

2016 ◽

Vol 3 ◽

pp. 16057 ◽

Cited By ~ 16

Author(s):

Arnold Park ◽

Patrick Hong ◽

Sohui T Won ◽

Patricia A Thibault ◽

Frederic Vigant ◽

...

Keyword(s):

Gene Editing ◽

Sendai Virus ◽

Rna Virus ◽

Genomic Integration ◽

Efficient Gene

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Advances in therapeutic application of CRISPR-Cas9

Briefings in Functional Genomics ◽

10.1093/bfgp/elz031 ◽

2019 ◽

Vol 19 (3) ◽

pp. 164-174 ◽

Cited By ~ 3

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.

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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.

10.1101/023366 ◽

2015 ◽

Cited By ~ 3

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.

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Optimization of T-DNA architecture for Cas9-mediated mutagenesis in Arabidopsis

10.1101/419952 ◽

2018 ◽

Author(s):

Baptiste Castel ◽

Laurence Tomlinson ◽

Federica Locci ◽

Ying Yang ◽

Jonathan D G Jones

Keyword(s):

Functional Analysis ◽

Gene Editing ◽

First Generation ◽

Gene Families ◽

Loss Of Function ◽

Site Specific ◽

Systematic Comparison ◽

Multiple Promoters ◽

Highly Active ◽

Homozygous Mutations

ABSTRACTBacterial CRISPR systems have been widely adopted to create operator-specified site-specific nucleases. Such nuclease action commonly results in loss-of-function alleles, facilitating functional analysis of genes and gene families We conducted a systematic comparison of components and T-DNA architectures for CRISPR-mediated gene editing in Arabidopsis, testing multiple promoters, terminators, sgRNA backbones and Cas9 alleles. We identified a T-DNA architecture that usually results in stable (i.e. homozygous) mutations in the first generation after transformation. Notably, the transcription of sgRNA and Cas9 in head-to-head divergent orientation usually resulted in highly active lines. Our Arabidopsis data may prove useful for optimization of CRISPR methods in other plants.

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Efficient Gene Editing Through an Intronic Selection Marker in Cells

10.21203/rs.3.rs-739001/v1 ◽

2021 ◽

Author(s):

Shang Wang ◽

Yuqing Li ◽

Li Zhong ◽

Kai Wu ◽

Ruhua Zhang ◽

...

Keyword(s):

Gene Editing ◽

Selection Marker ◽

End Joining ◽

Dna Variation ◽

Editing Efficiency ◽

Endogenous Gene ◽

Positive Effects ◽

Efficient Gene ◽

Almost All ◽

In Cells

Abstract Background Gene editing technology has provided researchers with the ability to modify genome sequences in almost all eukaryotes. Gene-edited cell lines are being used with increasing frequency in both bench research and targeted therapy. Despite the great importance and universality of gene editing, however, precision and efficiency are hard to achieve with the prevailing editing strategies, such as homology-directed DNA repair (HDR) and the use of base editors (BEs). Results & Discussion Our group has developed a novel gene editing technology to indicate DNA variation with an independent selection marker using an HDR strategy, which we named Gene Editing through an Intronic Selection marker (GEIS). GEIS uses a simple process to avoid nonhomologous end joining (NHEJ)-mediated false-positive effects and achieves editing efficiency as high as 91% without disturbing endogenous gene splicing and expression. We re-examined the correlation of the conversion tract and editing efficiency, and our data suggest that GEIS has the potential to edit approximately 99% of gene editing targets in human and mouse cells. The results of further comprehensive analysis suggest that the strategy may be useful for introducing multiple DNA variations in cells.

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