RNA silencing suppressor-influenced performance of a virus vector delivering both guide RNA and Cas9 for CRISPR gene editing

AbstractWe report on further development of the agroinfiltratable Tobacco mosaic virus (TMV)-based overexpression (TRBO) vector to deliver CRISPR/Cas9 components into plants. First, production of a Cas9 (HcoCas9) protein from a binary plasmid increased when co-expressed in presence of suppressors of gene silencing, such as the TMV 126-kDa replicase or the Tomato bushy stunt virus P19 protein. Such suppressor-generated elevated levels of Cas9 expression translated to efficient gene editing mediated by TRBO-G-3′gGFP expressing GFP and also a single guide RNA targeting the mgfp5 gene in the Nicotiana benthamiana GFP-expressing line 16c. Furthermore, HcoCas9 encoding RNA, a large cargo insert of 4.2 kb, was expressed from TRBO-HcoCas9 to yield Cas9 protein again at higher levels upon co-expression with P19. Likewise, co-delivery of TRBO-HcoCas9 and TRBO-G-3′gGFP in the presence of P19 also resulted in elevated levels percentages of indels (insertions and deletions). These data also revealed an age-related phenomenon in plants whereby the RNA suppressor P19 had more of an effect in older plants. Lastly, we used a single TRBO vector to express both Cas9 and a sgRNA. Taken together, we suggest that viral RNA suppressors could be used for further optimization of single viral vector delivery of CRISPR gene editing parts.

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Application of CRISPR/Cas9 technology in wild apple (Malus sieverii) for paired sites gene editing

Plant Methods ◽

10.1186/s13007-021-00769-8 ◽

2021 ◽

Vol 17 (1) ◽

Author(s):

Yan Zhang ◽

Ping Zhou ◽

Tohir A. Bozorov ◽

Daoyuan Zhang

Keyword(s):

Abiotic Stress ◽

Human Activities ◽

Genetic Improvement ◽

Gene Editing ◽

New Technologies ◽

Tianshan Mountains ◽

Biotic And Abiotic Stress ◽

Guide Rna ◽

Cas9 Protein ◽

Albino Phenotype

Abstract Background Xinjiang wild apple is an important tree of the Tianshan Mountains, and in recent years, it has undergone destruction by many biotic and abiotic stress and human activities. It is necessary to use new technologies to research its genomic function and molecular improvement. The clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system has been successfully applied to genetic improvement in many crops, but its editing capability varies depending on the different combinations of the synthetic guide RNA (sgRNA) and Cas9 protein expression devices. Results In this study, we used 2 systems of vectors with paired sgRNAs targeting to MsPDS. As expected, we successfully induced the albino phenotype of calli and buds in both systems. Conclusions We conclude that CRISPR/Cas9 is a powerful system for editing the wild apple genome and expands the range of plants available for gene editing.

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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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A Multiplex CRISPR/Cas9 System for Use as an Anti-BmNPV Therapeutic

10.20944/preprints201811.0159.v1 ◽

2018 ◽

Author(s):

Zhanqi Dong ◽

Qi Qin ◽

Zhigang Hu ◽

Peng Chen ◽

Liang Huang ◽

...

Keyword(s):

Gene Editing ◽

Genome Engineering ◽

Sustained Delivery ◽

Dna Breaks ◽

Guide Rna ◽

Cas9 Protein ◽

Transgenic Silkworms ◽

Enzyme Targeting ◽

Multiplex System ◽

Unique Capability

Clustered regularly interspaced short palindromic repeats/associated protein 9 nuclease (CRISPR/Cas9) technology guided by a single-guide RNA (sgRNA) has recently opened a new avenue for antiviral therapy. A unique capability of the CRISPR/Cas9 system is multiple genome engineering. However, there are few applications in insect viruses by a single Cas9 enzyme targeting two or more sgRNA at different genomic sites for simultaneous production of multiple DNA breaks. To address the need for multi-gene editing and sustained delivery of multiplex CRISPR/Cas9-based genome engineering tools, we developed a one-vector (pSL1180-Cas9-U6-sgRNA) system to express multiple sgRNA and Cas9 protein to excise Bombyx mori nucleopolyhedrovirus (BmNPV) in insect cells. Here, ie-1, gp64, lef-11, and dnapol genes were screened and identified as multiple sgRNA editing sites according to the BmNPV system infection and DNA replication mechanism. Furthermore, we constructed a multiplex editing vector sgMultiple to efficiently regulate multiplex gene editing steps and inhibit BmNPV replication after viral infection. This is the first report that describes the application of multiplex CRISPR/Cas9 system inhibiting insect virus replication. This multiplex system can significant enable the potential of CRISPR/Cas9-based multiplex genome engineering in transgenic silkworms.

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Various Aspects of a Gene Editing System—CRISPR–Cas9

International Journal of Molecular Sciences ◽

10.3390/ijms21249604 ◽

2020 ◽

Vol 21 (24) ◽

pp. 9604

Author(s):

Edyta Janik ◽

Marcin Niemcewicz ◽

Michal Ceremuga ◽

Lukasz Krzowski ◽

Joanna Saluk-Bijak ◽

...

Keyword(s):

Genome Editing ◽

Gene Editing ◽

Genome Engineering ◽

High Efficiency ◽

Adaptive Immune System ◽

Zinc Finger Nucleases ◽

Transcription Activator ◽

Guide Rna ◽

Adaptive Immune ◽

Cas9 Protein

The discovery of clustered, regularly interspaced short palindromic repeats (CRISPR) and their cooperation with CRISPR-associated (Cas) genes is one of the greatest advances of the century and has marked their application as a powerful genome engineering tool. The CRISPR–Cas system was discovered as a part of the adaptive immune system in bacteria and archaea to defend from plasmids and phages. CRISPR has been found to be an advanced alternative to zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN) for gene editing and regulation, as the CRISPR–Cas9 protein remains the same for various gene targets and just a short guide RNA sequence needs to be altered to redirect the site-specific cleavage. Due to its high efficiency and precision, the Cas9 protein derived from the type II CRISPR system has been found to have applications in many fields of science. Although CRISPR–Cas9 allows easy genome editing and has a number of benefits, we should not ignore the important ethical and biosafety issues. Moreover, any tool that has great potential and offers significant capabilities carries a level of risk of being used for non-legal purposes. In this review, we present a brief history and mechanism of the CRISPR–Cas9 system. We also describe on the applications of this technology in gene regulation and genome editing; the treatment of cancer and other diseases; and limitations and concerns of the use of CRISPR–Cas9.

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Somatic cell reprogramming-free generation of genetically modified pigs

Science Advances ◽

10.1126/sciadv.1600803 ◽

2016 ◽

Vol 2 (9) ◽

pp. e1600803 ◽

Cited By ~ 40

Author(s):

Fuminori Tanihara ◽

Tatsuya Takemoto ◽

Eri Kitagawa ◽

Shengbin Rao ◽

Lanh Thi Kim Do ◽

...

Keyword(s):

Somatic Cell ◽

Gene Editing ◽

Genetically Modified ◽

Efficient Production ◽

Simple Method ◽

Guide Rna ◽

Somatic Cell Reprogramming ◽

Cas9 Protein ◽

Somatic Cell Nucleus

Genetically modified pigs for biomedical applications have been mainly generated using the somatic cell nuclear transfer technique; however, this approach requires complex micromanipulation techniques and sometimes increases the risks of both prenatal and postnatal death by faulty epigenetic reprogramming of a donor somatic cell nucleus. As a result, the production of genetically modified pigs has not been widely applied. We provide a simple method for CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 gene editing in pigs that involves the introduction of Cas9 protein and single-guide RNA into in vitro fertilized zygotes by electroporation. The use of gene editing by electroporation of Cas9 protein (GEEP) resulted in highly efficient targeted gene disruption and was validated by the efficient production of Myostatin mutant pigs. Because GEEP does not require the complex methods associated with micromanipulation for somatic reprogramming, it has the potential for facilitating the genetic modification of pigs.

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Disabling Cas9 by an anti-CRISPR DNA mimic

10.1101/129627 ◽

2017 ◽

Cited By ~ 5

Author(s):

Jiyung Shing ◽

Fuguo Jiang ◽

Jun-Jie Liu ◽

Nicholas L. Bray ◽

Benjamin J. Rauch ◽

...

Keyword(s):

Mammalian Cells ◽

Gene Editing ◽

Adaptive Immune System ◽

Binding Mode ◽

Human Cells ◽

Guide Rna ◽

Protospacer Adjacent Motif ◽

Cas9 Protein ◽

Potential Applications ◽

Natural Inhibitors

CRISPR-Cas9 gene editing technology is derived from a microbial adaptive immune system, where bacteriophages are often the intended target. Natural inhibitors of CRISPR-Cas9 enable phages to evade immunity and show promise in controlling Cas9-mediated gene editing in human cells. However, the mechanism of CRISPR-Cas9 inhibition is not known and the potential applications for Cas9 inhibitor proteins in mammalian cells has not fully been established. We show here that the anti-CRISPR protein AcrIIA4 binds only to assembled Cas9-single guide RNA (sgRNA) complexes and not to Cas9 protein alone. A 3.9 Å resolution cryo-EM structure of the Cas9-sgRNA-AcrIIA4 complex revealed that the surface of AcrIIA4 is highly acidic and binds with 1:1 stoichiometry to a region of Cas9 that normally engages the DNA protospacer adjacent motif (PAM). Consistent with this binding mode, order-of-addition experiments showed that AcrIIA4 interferes with DNA recognition but has no effect on pre-formed Cas9-sgRNA-DNA complexes. Timed delivery of AcrIIA4 into human cells as either protein or expression plasmid allows on-target Cas9-mediated gene editing while reducing off-target edits. These results provide a mechanistic understanding of AcrIIA4 function and demonstrate that inhibitors can modulate the extent and outcomes of Cas9-mediated gene editing.

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CRISPR Systems Suitable for Single AAV Vector Delivery

Current Gene Therapy ◽

10.2174/1566523221666211006120355 ◽

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.

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

Journal of Fungi ◽

10.3390/jof7070505 ◽

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.

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Improved Transformation and Regeneration of Indica Rice: Disruption of SUB1A as a Test Case via CRISPR-Cas9

International Journal of Molecular Sciences ◽

10.3390/ijms22136989 ◽

2021 ◽

Vol 22 (13) ◽

pp. 6989

Author(s):

Yuya Liang ◽

Sudip Biswas ◽

Backki Kim ◽

Julia Bailey-Serres ◽

Endang M. Septiningsih

Keyword(s):

Gene Editing ◽

Indica Rice ◽

Crop Improvement ◽

Immature Embryo ◽

Test Case ◽

Submergence Tolerance ◽

Embryogenic Calli ◽

Submergence Stress ◽

Efficient Vector ◽

Vector Delivery

Gene editing by use of clustered regularly interspaced short palindromic repeats (CRISPR) has become a powerful tool for crop improvement. However, a common bottleneck in the application of this approach to grain crops, including rice (Oryza sativa), is efficient vector delivery and calli regeneration, which can be hampered by genotype-dependent requirements for plant regeneration. Here, methods for Agrobacterium-mediated and biolistic transformation and regeneration of indica rice were optimized using CRISPR-Cas9 gene-editing of the submergence tolerance regulator SUBMERGENCE 1A-1 gene of the cultivar Ciherang-Sub1. Callus induction and plantlet regeneration methods were optimized for embryogenic calli derived from immature embryos and mature seed-derived calli. Optimized regeneration (95%) and maximal editing efficiency (100%) were obtained from the immature embryo-derived calli. Phenotyping of T1 seeds derived from the edited T0 plants under submergence stress demonstrated inferior phenotype compared to their controls, which phenotypically validates the disruption of SUB1A-1 function. The methods pave the way for rapid CRISPR-Cas9 gene editing of recalcitrant indica rice cultivars.

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Non-viral delivery of CRISPR–Cas9 complexes for targeted gene editing via a polymer delivery system

Gene Therapy ◽

10.1038/s41434-021-00282-6 ◽

2021 ◽

Author(s):

Jonathan O’Keeffe Ahern ◽

Irene Lara-Sáez ◽

Dezhong Zhou ◽

Rodolfo Murillas ◽

Jose Bonafont ◽

...

Keyword(s):

Delivery System ◽

Gene Editing ◽

Transfection Efficiency ◽

Attractive Alternative ◽

Safe Delivery ◽

Guide Rna ◽

Recessive Dystrophic Epidermolysis Bullosa ◽

Targeted Gene Editing ◽

Genomic Editing ◽

Polymeric Vectors

AbstractRecent advances in molecular biology have led to the CRISPR revolution, but the lack of an efficient and safe delivery system into cells and tissues continues to hinder clinical translation of CRISPR approaches. Polymeric vectors offer an attractive alternative to viruses as delivery vectors due to their large packaging capacity and safety profile. In this paper, we have demonstrated the potential use of a highly branched poly(β-amino ester) polymer, HPAE-EB, to enable genomic editing via CRISPRCas9-targeted genomic excision of exon 80 in the COL7A1 gene, through a dual-guide RNA sequence system. The biophysical properties of HPAE-EB were screened in a human embryonic 293 cell line (HEK293), to elucidate optimal conditions for efficient and cytocompatible delivery of a DNA construct encoding Cas9 along with two RNA guides, obtaining 15–20% target genomic excision. When translated to human recessive dystrophic epidermolysis bullosa (RDEB) keratinocytes, transfection efficiency and targeted genomic excision dropped. However, upon delivery of CRISPR–Cas9 as a ribonucleoprotein complex, targeted genomic deletion of exon 80 was increased to over 40%. Our study provides renewed perspective for the further development of polymer delivery systems for application in the gene editing field in general, and specifically for the treatment of RDEB.

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