Cas13b-dependent and Cas13b-independent RNA knockdown of viral sequences in mosquito cells following guide RNA expression

AbstractAedes aegypti and Aedes albopictus mosquitoes are vectors of the RNA viruses chikungunya (CHIKV) and dengue that currently have no specific therapeutic treatments. The development of new methods to generate virus-refractory mosquitoes would be beneficial. Cas13b is an enzyme that uses RNA guides to target and cleave RNA molecules and has been reported to suppress RNA viruses in mammalian and plant cells. We investigated the potential use of the Prevotella sp. P5-125 Cas13b system to provide viral refractoriness in mosquito cells, using a virus-derived reporter and a CHIKV split replication system. Cas13b in combination with suitable guide RNAs could induce strong suppression of virus-derived reporter RNAs in insect cells. Surprisingly, the RNA guides alone (without Cas13b) also gave substantial suppression. Our study provides support for the potential use of Cas13b in mosquitoes, but also caution in interpreting CRISPR/Cas data as we show that guide RNAs can have Cas-independent effects.

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A viral guide RNA delivery system for CRISPR-based transcriptional activation and heritable targeted DNA demethylation in Arabidopsis thaliana

10.1101/2020.07.09.194977 ◽

2020 ◽

Author(s):

Basudev Ghoshal ◽

Brandon Vong ◽

Colette L. Picard ◽

Feng Suhua ◽

Janet May Tam ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

Genome Editing ◽

Delivery System ◽

Transcriptional Activation ◽

Rna Viruses ◽

Tobacco Rattle Virus ◽

Dna Demethylation ◽

Guide Rna ◽

Epigenome Editing ◽

Guide Rnas

AbstractPlant RNA viruses are used as delivery vectors for their high level of accumulation and efficient spread during virus multiplication and movement. Utilizing this concept, several viral-based guide RNA delivery platforms for CRISPR-Cas9 genome editing have been developed. The CRISPR-Cas9 system has also been adapted for epigenome editing. While systems have been developed for CRISPR-Cas9 based gene activation or site-specific DNA demethylation, viral delivery of guide RNAs remains to be developed for these purposes. To address this gap we have developed a tobacco rattle virus (TRV)-based single guide RNA delivery system for epigenome editing in Arabidopsis thaliana. Because tRNA-like sequences have been shown to facilitate the cell-to-cell movement of RNAs in plants, we used the tRNA-guide RNA expression system to express guide RNAs from the viral genome to promote heritable epigenome editing. We demonstrate that the tRNA-gRNA system with TRV can be used for both transcriptional activation and targeted DNA demethylation in Arabidopsis. We achieved up to ~8% heritability of the induced demethylation phenotype in the progeny of virus inoculated plants. We did not detect the virus in the next generation, indicating effective clearance of the virus from plant tissues. Thus, TRV delivery, combined with a specific tRNA-gRNA architecture, provides for fast and effective epigenome editing.Author summaryThe discovery of CRISPR-CAS9 and its non-catalytic variants have provided enormous capacity for crop improvement and basic research by modifying the genome and the epigenome. The standard methods for delivering genome and epigenome editing reagents to plants consist of generating stable transgenic lines through tissue culture processes, which have several drawbacks including the need for plant regeneration and crossing. To overcome some of these challenges, plant virus-based platforms are being developed for genome editing. Although viruses have a limited cargo capacity, limiting the use of viruses to encode entire editing systems, guide RNAs have been successfully delivered to transgenic CAS9 expressing plants for genome editing. However, the use of viruses for CRISPR-based epigenome editing and transcriptional activation have not yet been explored. In this study we show that viral delivery of guide RNAs using a modified tobacco rattle virus can be used for transcriptional activation and heritable epigenome editing. This study advances the use of plant RNA viruses as delivery agents for epigenome editing.

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Investigation of the properties and activity of DfCas9 and DsCas9 nucleases in eucaryotic cells

Pharmacy Formulas ◽

10.17816/phf80850 ◽

2021 ◽

Author(s):

Yelizaveta V. Vlasova ◽

Dmitry A. Madera ◽

Pavel M. Gershovich

Keyword(s):

Protein Detection ◽

Nuclease Activity ◽

Hek293 Cells ◽

Rna Molecules ◽

Guide Rna ◽

Guide Rnas ◽

Cloning Method ◽

Science And Education ◽

Genetic Constructs

This study is focused on the two novel nucleases of the CRISPR/Cas9 family, which were found in bacterial genomes of DfCas9 (Defluviimonas sp) и DsCas9 (Demequina sediminicola). Discovery of these nucleases was part of the results of a joint study conducted by BIOCAD together with Skoltech Institute of Science and Technology and Saint-Petersburg Polytechnical University (SPPU) under a grant agreement with the Department of Science and Education of Russian Federation (Agreement number 14.606.21.0006 from September, 26th 2017). Under the agreement the nucleases DfCas9 and DsCas9 were characterized in vitro by Skoltech and SPPU. Based on the aforementioned results, in this study we characterized the genome-modifying nuclease activity of these enzymes in a mammalian cell line HEK293. Specifically, we created genetic constructs designed to express the nucleases DsCas9 and DfCas9 together with the necessary guide RNA molecules (sequences of the guide RNAs were described previously) [1]. We demonstrated expression of the nucleases on a protein level, as well as activity of DfCas9 at the VEGF2 locus in HEK293 cells. The theoretical study was conducted by analyzing international and national literature. The experimental part was performed with a restriction-ligation cloning method, transient transfections, Western blot protein detection method, and a T7 nuclease-based method of detection of heteroduplex double-stranded DNA.

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ASSESSMENT OF EFFICIENCY AND OFF-TARGET ACTIVITY OF CRISPR/CAS RIBONUCLEOPROTEIN COMPLEXES

Molecular Diagnostics and Biosafety – 2020. Russian national scientific and practical conference with international participation (October, 6–8, 2020): Conference Proceedings ◽

10.36233/978-5-9900432-9-9-98 ◽

2020 ◽

Author(s):

Y.V. Mikhaylova ◽

◽

M.A. Tyumentseva ◽

A.A. Shelenkov ◽

Y.G. Yanushevich ◽

...

Keyword(s):

High Sensitivity ◽

Correct Choice ◽

Target Sequence ◽

Dna Breaks ◽

Gene Encoding ◽

Guide Rna ◽

Guide Rnas ◽

Ribonucleoprotein Complexes ◽

Chemokine Receptor Ccr5 ◽

Target Activity

In this study, we assessed the efficiency and off-target activity of the CRISPR/CAS complex with one of the selected guide RNAs using the CIRCLE-seq technology. The gene encoding the human chemokine receptor CCR5 was used as a target sequence for genome editing. The results of this experiment indicate the correct choice of the guide RNA and efficient work of the CRISPR- CAS ribonucleoprotein complex used. CIRCLE-seq technology has shown high sensitivity compared to bioinformatic methods for predicting off-target activity of CRISPR/CAS complexes. We plan to evaluate the efficiency and off-target activity of CRISPR/CAS ribonucleoprotein complexes with other guide RNAs by slightly adjusting the CIRCLE-seq-technology protocol in order to reduce nonspecific DNA breaks and increase the number of reliable reads.

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An improved method for precise genome editing in zebrafish using CRISPR-Cas9 technique

Molecular Biology Reports ◽

10.1007/s11033-020-06125-8 ◽

2021 ◽

Author(s):

Eugene V. Gasanov ◽

Justyna Jędrychowska ◽

Michal Pastor ◽

Malgorzata Wiweger ◽

Axel Methner ◽

...

Keyword(s):

Genome Editing ◽

High Efficiency ◽

Target Site ◽

Proof Of Concept ◽

Intervention Site ◽

End Joining ◽

Guide Rna ◽

Guide Rnas ◽

Cas9 Nuclease ◽

Non Homologous End Joining

AbstractCurrent methods of CRISPR-Cas9-mediated site-specific mutagenesis create deletions and small insertions at the target site which are repaired by imprecise non-homologous end-joining. Targeting of the Cas9 nuclease relies on a short guide RNA (gRNA) corresponding to the genome sequence approximately at the intended site of intervention. We here propose an improved version of CRISPR-Cas9 genome editing that relies on two complementary guide RNAs instead of one. Two guide RNAs delimit the intervention site and allow the precise deletion of several nucleotides at the target site. As proof of concept, we generated heterozygous deletion mutants of the kcng4b, gdap1, and ghitm genes in the zebrafish Danio rerio using this method. A further analysis by high-resolution DNA melting demonstrated a high efficiency and a low background of unpredicted mutations. The use of two complementary gRNAs improves CRISPR-Cas9 specificity and allows the creation of predictable and precise mutations in the genome of D. rerio.

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Trimeric Hantavirus Nucleocapsid Protein Binds Specifically to the Viral RNA Panhandle

Journal of Virology ◽

10.1128/jvi.78.15.8281-8288.2004 ◽

2004 ◽

Vol 78 (15) ◽

pp. 8281-8288 ◽

Cited By ~ 53

Author(s):

M. A. Mir ◽

A. T. Panganiban

Keyword(s):

Nucleocapsid Protein ◽

Rna Binding ◽

Rna Viruses ◽

Sin Nombre Virus ◽

Viral Capsid ◽

Genomic Rna ◽

Rna Molecules ◽

N Protein ◽

High Affinity ◽

Negative Sense

ABSTRACT Hantaviruses are tripartite negative-sense RNA viruses and members of the Bunyaviridae family. The nucleocapsid (N) protein is encoded by the smallest of the three genome segments (S). N protein is the principal structural component of the viral capsid and is central to the hantavirus replication cycle. We examined intermolecular N-protein interaction and RNA binding by using bacterially expressed Sin Nombre virus N protein. N assembles into di- and trimeric forms. The mono- and dimeric forms exist transiently and assemble into a trimeric form. In contrast, the trimer is highly stable and does not efficiently disassemble into the mono- and dimeric forms. The purified N-protein trimer is able to discriminate between viral and nonviral RNA molecules and, interestingly, recognizes and binds with high affinity the panhandle structure composed of the 3′ and 5′ ends of the genomic RNA. In contrast, the mono- and dimeric forms of N bind RNA to form a complex that is semispecific and salt sensitive. We suggest that trimerization of N protein is a molecular switch to generate a protein complex that can discriminate between viral and nonviral RNA molecules during the early steps of the encapsidation process.

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CRISPR/Cas9 mediated knockout of the DES gene alleles with desminopathy-related heterozygous gain-of-function mutations

Nauchno-prakticheskii zhurnal «Medicinskaia genetika» ◽

10.25557/2073-7998.2021.07.37-44 ◽

2021 ◽

pp. 37-44

Author(s):

К.С. Кочергин-Никитский ◽

А.В. Лавров ◽

Е.В. Заклязьминская ◽

С.А. Смирнихина

Keyword(s):

Genome Editing ◽

Genetic Defect ◽

Gene Mutations ◽

Respiratory Muscles ◽

Surgical Approaches ◽

Nonsense Mutations ◽

Guide Rna ◽

Guide Rnas ◽

Patient State ◽

Targeted Nucleases

Наследственные кардиомиопатии характеризуются неблагоприятным прогнозом и низкой пятилетней выживаемостью пациентов с выраженной клиникой. При этом лечение, за исключением хирургического, в основном паллиативное, во многих случаях лишь трансплантация сердца может улучшить состояние пациента и прогноз. Часть наследственных кардиомиопатий ассоциирована с аутосомно-доминантными мутациями в гене DES, кодирующем белок промежуточных филаментов десмин, дефекты в котором ведут к развитию десминопатий с вовлечением наиболее активно работающих мышц - скелетных, миокарда, мышц дыхательной системы. Новые терапевтические подходы, основанные на методах геномного редактирования, могут позволить устранить каузативный генетический дефект. Так как имеются данные об отсутствии клинических симптомов у людей с гетерозиготными нонсенс мутациями в гене DES, по-видимому, имеется возможность снизить тяжесть протекания десминопатий путем нокаута мутантного аллеля в случае гетерозиготной мутации. Целью работы являлась проверка возможности специфического нокаута аллелей гена DES, несущих гетерозиготные мутации, ассоциированные с десминопатиями, методами геномного редактирования. Нами был получен генетический материал трех пациентов с десминопатиями, связанными с мутациями в гене DES (c.330_338del, p.A337P (c.1009G>C) и p.R355P (c.1064G>C)). Направляющие РНК, совместимые с нуклеазами SaCas9 и eSpCas9(1.1), были подобраны, используя онлайн сервис Benchling, и клонированы в плазмиды, несущие соответствующие эндонуклеазы Cas9. Редактирующие плазмиды котрансфицировали в клетки HEK293T вместе с «таргетными» плазмидами, содержащими участки гена DES с мутациями. Анализ характерных для негомологичного соединения концов инделов в выделенной из клеток спустя 48 часов после трансфекции тотальной ДНК проводился посредством TIDE-анализа полученных сиквенсов целевых участков, либо методом Т7Е1 анализа. Наибольшая средняя эффективность 2,22% (до 8,06%) показана при использовании sgRNA на мутацию c.330_338del в комбинации с eSpCas9(1.1). Эффективность других комбинаций направляющих РНК и Cas9 не превышала 3%. Достигнутая эффективность нокаута очевидно недостаточна для коррекции десминопатии на уровне организма. Необходимость специфического нокаутирования мутантных аллелей не позволяет использовать другие направляющие РНК для CRISPR/Cas9, поэтому необходимо совершенствование разработанных систем для повышения их эффективности либо использование новых, более эффективных, направляемых нуклеаз. Hereditary cardiomyopathies are characterized by the generally poor prognosis and low 5-year survival of patients with severe symptoms. Besides surgical approaches, cardiomyopathy therapy mainly palliative and often heart transplantation is the only option to improve patient state and prognosis. Some of these pathologies are associated with the autosomal-dominant DES gene mutations. DES encodes intermediate filaments protein desmin, which defects causes desminopathies involving most active muscles such as skeletal muscles, myocardium and respiratory muscles. New therapeutic based on genome editing approaches could be used to correct causative genetic defect. There are data that heterozygous nonsense mutations in DES gene may be asymptomatic. Thus there is, apparently, a possibility to decrease severity of desminopathy using mutant allele knockout. Purpose. The aim of this work was to test the possibility of specific knockout of the DES gene alleles with heterozygous desminopathy-associated mutations by means of genome editing methods. Materials. We received genetic materials of three patients with desminopathy caused by DES gene mutations (c.330_338del, p.A337P (c.1009G>C) и p.R355P (c.1064G>C)). Guide RNA, compatible with nucleases SaCas9 and eSpCas9(1.1) were designed using online service Benchling and cloned into plasmids with corresponding Cas9 nucleases. Editing plasmids were cotransfected into HEK293T cells with “target” plasmids, containing DES gene sites with mutations. NHEJ-produced indels were assessed using TIDE-analysis with amplified and sequenced sites or using T7E1 analysis. Results. Combination sgRNA for c.330_338del with eSpCas9(1.1) demonstrated most mean efficiency of 2,22% (up to 8,06%). Others combinations of sgRNAs and Cas9 efficiency did not overcome 3%. Conclusions. Achieved knockout efficiency is evidently not enough for organism-level desminopathy correction. The need for specific knockout of mutated alleles does not allow usage of different guide RNAs for CRISPR/Cas9, so it is necessary to improve the developed systems to increase their efficiency or to use new, more efficient, targeted nucleases.

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Circumventing Zygotic Lethality to Generate Maternal Mutants in Zebrafish

Biology ◽

10.3390/biology11010102 ◽

2022 ◽

Vol 11 (1) ◽

pp. 102

Author(s):

De-Li Shi

Keyword(s):

Germ Line ◽

Gene Products ◽

Transgenic Expression ◽

Guide Rna ◽

Guide Rnas ◽

Transgenic Embryos ◽

Mutant Phenotypes ◽

Biallelic Mutations ◽

Genome Activation ◽

Maternal Gene

Maternal gene products accumulated during oogenesis are essential for supporting early developmental processes in both invertebrates and vertebrates. Therefore, understanding their regulatory functions should provide insights into the maternal control of embryogenesis. The CRISPR/Cas9 genome editing technology has provided a powerful tool for creating genetic mutations to study gene functions and developing disease models to identify new therapeutics. However, many maternal genes are also essential after zygotic genome activation; as a result, loss of their zygotic functions often leads to lethality or sterility, thus preventing the generation of maternal mutants by classical crossing between zygotic homozygous mutant adult animals. Although several approaches, such as the rescue of mutant phenotypes through an injection of the wild-type mRNA, germ-line replacement, and the generation of genetically mosaic females, have been developed to overcome this difficulty, they are often technically challenging and time-consuming or inappropriate for many genes that are essential for late developmental events or for germ-line formation. Recently, a method based on the oocyte transgenic expression of CRISPR/Cas9 and guide RNAs has been designed to eliminate maternal gene products in zebrafish. This approach introduces several tandem guide RNA expression cassettes and a GFP reporter into transgenic embryos expressing Cas9 to create biallelic mutations and inactivate genes of interest specifically in the developing oocytes. It is particularly accessible and allows for the elimination of maternal gene products in one fish generation. By further improving its efficiency, this method can be used for the systematic characterization of maternal-effect genes.

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A modular cloning toolkit for genome editing in plants

10.1101/738021 ◽

2019 ◽

Cited By ~ 2

Author(s):

Florian Hahn ◽

Andrey Korolev ◽

Laura Sanjurjo Loures ◽

Vladimir Nekrasov

Keyword(s):

Genome Editing ◽

Higher Order ◽

Plant Science ◽

Golden Gate ◽

Coding Sequences ◽

Guide Rna ◽

Guide Rnas ◽

Science Community ◽

Modular Cloning ◽

Multiple Expression

AbstractBackgroundCRISPR/Cas has recently become a widely used genome editing tool in various organisms, including plants. Applying CRISPR/Cas often requires delivering multiple expression units into plant and hence there is a need for a quick and easy cloning procedure. The modular cloning (MoClo), based on the Golden Gate (GG) method, has enabled development of cloning systems with standardised genetic parts, e.g. promoters, coding sequences or terminators, that can be easily interchanged and assembled into expression units, which in their own turn can be further assembled into higher order multigene constructs.ResultsHere we present an expanded cloning toolkit that contains ninety-nine modules encoding a variety of CRISPR/Cas-based nucleases and their corresponding guide RNA backbones. Among other components, the toolkit includes a number of promoters that allow expression of CRISPR/Cas nucleases (or any other coding sequences) and their guide RNAs in monocots and dicots. As part of the toolkit, we present a set of modules that enable quick and facile assembly of tRNA-sgRNA polycistronic units without a PCR step involved. We also demonstrate that our tRNA-sgRNA system is functional in wheat protoplasts.ConclusionsWe believe the presented CRISPR/Cas toolkit is a great resource that will contribute towards wider adoption of the CRISPR/Cas genome editing technology and modular cloning by researchers across the plant science community.

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Improving the Precision of Base Editing by Bubble Hairpin Single Guide RNA

mBio ◽

10.1128/mbio.00342-21 ◽

2021 ◽

Vol 12 (2) ◽

Author(s):

Zhiwei Hu ◽

Yannan Wang ◽

Qian Liu ◽

Yan Qiu ◽

Zhiyu Zhong ◽

...

Keyword(s):

Genome Editing ◽

Dna Breaks ◽

Single Nucleotide ◽

Base Editing ◽

Guide Rna ◽

Guide Rnas ◽

Double Stranded Dna ◽

Target Sites ◽

Guide Sequence ◽

Sgrna Design

ABSTRACT Base editing is a powerful genome editing approach that enables single-nucleotide changes without double-stranded DNA breaks (DSBs). However, off-target effects as well as other undesired editings at on-target sites remain obstacles for its application. Here, we report that bubble hairpin single guide RNAs (BH-sgRNAs), which contain a hairpin structure with a bubble region on the 5′ end of the guide sequence, can be efficiently applied to both cytosine base editor (CBE) and adenine base editor (ABE) and significantly decrease off-target editing without sacrificing on-target editing efficiency. Meanwhile, such a design also improves the purity of C-to-T conversions induced by base editor 3 (BE3) at on-target sites. Our results present a distinctive and effective strategy to improve the specificity of base editing. IMPORTANCE Base editors are DSB-free genome editing tools and have been widely used in diverse living systems. However, it is reported that these tools can cause substantial off-target editings. To meet this challenge, we developed a new approach to improve the specificity of base editors by using hairpin sgRNAs with a bubble. Furthermore, our sgRNA design also dramatically reduced indels and unwanted base substitutions at on-target sites. We believe that the BH-sgRNA design is a significant improvement over existing sgRNAs of base editors, and our design promises to be adaptable to various base editors. We expect that it will make contributions to improving the safety of gene therapy.

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Trypanosoma equiperdum minicircles encode three distinct primary transcripts which exhibit guide RNA characteristics

Molecular and Cellular Biology ◽

10.1128/mcb.11.3.1668-1675.1991 ◽

1991 ◽

Vol 11 (3) ◽

pp. 1668-1675

Author(s):

V W Pollard ◽

S L Hajduk

Keyword(s):

Size Range ◽

Transcription Unit ◽

Guide Rna ◽

Conserved Sequence ◽

Guide Rnas ◽

Sequence Class ◽

Trypanosoma Equiperdum ◽

Transcript Initiation ◽

Size Heterogeneity ◽

Single Sequence

The mitochondrial DNA of trypanosomes is composed of maxicircle and minicircle DNAs catenated into a network, called the kinetoplast. Maxicircles encode proteins and RNAs necessary for mitochondrial assembly. Minicircles encode small transcripts which are believed to serve as guide RNAs in the process of RNA editing of maxicircle transcripts. Trypanosoma equiperdum minicircles contain three transcription units which produce three distinct transcripts. The genes for these transcripts are flanked by imperfect 18-bp repeats separated by approximately 110 bp. The transcripts have a 5' triphosphate, indicating that they are primary transcripts. Minicircle transcription initiates at a purine within a conserved sequence, 5'-AYAYA-3', where Y is a pyrimidine, 32 bp from the upstream inverted repeat, suggesting that the repeats may function in transcript initiation. Transcripts from a single minicircle transcription unit range in size from 55 to 70 nucleotides. This size heterogeneity within a single sequence class is due to the variable length of nontemplated uridine residues composing a 3' tail. The size range and heterogeneous polyuridylate 3' end of the minicircle transcripts appear to be conserved features and may be related to transcript function.

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