scholarly journals A modular cloning toolkit for genome editing in plants

2019 ◽  
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.

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

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.

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

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.

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

mBio ◽  
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.

scholarly journals kRISP-meR: A Reference-free Guide-RNA Design Tool for CRISPR/Cas9

2019 ◽  
Author(s):  
Mahmudur Rahman Hera ◽  
Amatur Rahman ◽  
Atif Rahman
Keyword(s):  
Genome Editing ◽  
Design Tool ◽  
Target Region ◽  
Guide Rna ◽  
Guide Rnas ◽  
Rna Design ◽  
Reference Genomes ◽  
Target Effects

AbstractGenome editing using the CRISPR/Cas9 system requires designing guide RNAs (sgRNA) that are efficient and specific. Guide RNAs are usually designed using reference genomes which limits their use in organisms with no or incomplete reference genomes. Here, we present kRISP-meR, a reference free method to design sgRNAs for CRISPR/Cas9 system. kRISP-meR takes as input a target region and sequenced reads from the organism to be edited and generates sgRNAs that are likely to minimize off-target effects. Our analysis indicates that kRISP-meR is able to identify majority of the guides identified by a widely used sgRNA designing tool, without any knowledge of the reference, while retaining specificity.

scholarly journals Development of a Csy4-processed guide RNA delivery system with soybean-infecting virus ALSV for genome editing

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Yanjie Luo ◽  
Ren Na ◽  
Julia S. Nowak ◽  
Yang Qiu ◽  
Qing Shi Lu ◽  
...  
Keyword(s):  
Genome Editing ◽  
Delivery System ◽  
Gene Function ◽  
Function Analysis ◽  
Soybean Plant ◽  
Apple Latent Spherical Virus ◽  
Guide Rna ◽  
Guide Rnas ◽  
Plant Trait ◽  
Trait Improvement

Abstract Background A key issue for implementation of CRISPR-Cas9 genome editing for plant trait improvement and gene function analysis is to efficiently deliver the components, including guide RNAs (gRNAs) and Cas9, into plants. Plant virus-based gRNA delivery strategy has proven to be an important tool for genome editing. However, its application in soybean which is an important crop has not been reported yet. ALSV (apple latent spherical virus) is highly infectious virus and could be explored for delivering elements for genome editing. Results To develop a ALSV-based gRNA delivery system, the Cas9-based Csy4-processed ALSV Carry (CCAC) system was developed. In this system, we engineered the soybean-infecting ALSV to carry and deliver gRNA(s). The endoribonuclease Csy4 effectively releases gRNAs that function efficiently in Cas9-mediated genome editing. Genome editing of endogenous phytoene desaturase (PDS) loci and exogenous 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) sequence in Nicotiana. benthamiana (N. benthamiana) through CCAC was confirmed using Sanger sequencing. Furthermore, CCAC-induced mutagenesis in two soybean endogenous GW2 paralogs was detected. Conclusions With the aid of the CCAC system, the target-specific gRNA(s) can be easily manipulated and efficiently delivered into soybean plant cells by viral infection. This is the first virus-based gRNA delivery system for soybean for genome editing and can be used for gene function study and trait improvement.

scholarly journals Adenovirus Vectors Expressing Eight Multiplex Guide RNAs of CRISPR/Cas9 Efficiently Disrupted Diverse Hepatitis B Virus Gene Derived from Heterogeneous Patient

2021 ◽  
Vol 22 (19) ◽  
pp. 10570
Author(s):  
Yuya Kato ◽  
Hirotaka Tabata ◽  
Kumiko Sato ◽  
Mariko Nakamura ◽  
Izumu Saito ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Genome Editing ◽  
Adenovirus Vector ◽  
Target Sequence ◽  
Guide Rna ◽  
Guide Rnas ◽  
B Virus ◽  
X Gene

Hepatitis B virus (HBV) chronically infects more than 240 million people worldwide, causing chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). Genome editing using CRISPR/Cas9 could provide new therapies because it can directly disrupt HBV genomes. However, because HBV genome sequences are highly diverse, the identical target sequence of guide RNA (gRNA), 20 nucleotides in length, is not necessarily present intact in the target HBV DNA in heterogeneous patients. Consequently, possible genome-editing drugs would be effective only for limited numbers of patients. Here, we show that an adenovirus vector (AdV) bearing eight multiplex gRNA expression units could be constructed in one step and amplified to a level sufficient for in vivo study with lack of deletion. Using this AdV, HBV X gene integrated in HepG2 cell chromosome derived from a heterogeneous patient was cleaved at multiple sites and disrupted. Indeed, four targets out of eight could not be cleaved due to sequence mismatches, but the remaining four targets were cleaved, producing irreversible deletions. Accordingly, the diverse X gene was disrupted at more than 90% efficiency. AdV containing eight multiplex gRNA units not only offers multiple knockouts of genes, but could also solve the problems of heterogeneous targets and escape mutants in genome-editing therapy.

scholarly journals Quantification of the affinities of CRISPR–Cas9 nucleases for cognate protospacer adjacent motif (PAM) sequences

2020 ◽  
Vol 295 (19) ◽  
pp. 6509-6517 ◽  
Author(s):  
Vladimir Mekler ◽  
Konstantin Kuznedelov ◽  
Konstantin Severinov
Keyword(s):  
Genome Editing ◽  
Target Location ◽  
Dna Probes ◽  
Target Sequence ◽  
Francisella Novicida ◽  
Guide Rna ◽  
Guide Rnas ◽  
Cas9 Nuclease ◽  
Protospacer Adjacent Motif ◽  
Target Dna

The CRISPR/Cas9 nucleases have been widely applied for genome editing in various organisms. Cas9 nucleases complexed with a guide RNA (Cas9–gRNA) find their targets by scanning and interrogating the genomic DNA for sequences complementary to the gRNA. Recognition of the DNA target sequence requires a short protospacer adjacent motif (PAM) located outside this sequence. Given that the efficiency of target location may depend on the strength of interactions that promote target recognition, here we sought to compare affinities of different Cas9 nucleases for their cognate PAM sequences. To this end, we measured affinities of Cas9 nucleases from Streptococcus pyogenes, Staphylococcus aureus, and Francisella novicida complexed with guide RNAs (gRNAs) (SpCas9–gRNA, SaCas9–gRNA, and FnCas9–gRNA, respectively) and of three engineered SpCas9–gRNA variants with altered PAM specificities for short, PAM-containing DNA probes. We used a “beacon” assay that measures the relative affinities of DNA probes by determining their ability to competitively affect the rate of Cas9–gRNA binding to fluorescently labeled target DNA derivatives called “Cas9 beacons.” We observed significant differences in the affinities for cognate PAM sequences among the studied Cas9 enzymes. The relative affinities of SpCas9–gRNA and its engineered variants for canonical and suboptimal PAMs correlated with previous findings on the efficiency of these PAM sequences in genome editing. These findings suggest that high affinity of a Cas9 nuclease for its cognate PAM promotes higher genome-editing efficiency.

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

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.

scholarly journals Using Synthetically Engineered Guide RNAs to Enhance CRISPR Genome Editing Systems in Mammalian Cells

2021 ◽  
Vol 2 ◽  
Author(s):  
Daniel Allen ◽  
Michael Rosenberg ◽  
Ayal Hendel
Keyword(s):  
Genome Editing ◽  
Mammalian Cells ◽  
Nuclear Staining ◽  
Two Component System ◽  
Guide Rna ◽  
Homology Directed Repair ◽  
Guide Rnas ◽  
Therapeutic Benefits ◽  
A Genome ◽  
Cas9 Protein

CRISPR-Cas9 is quickly revolutionizing the way we approach gene therapy. CRISPR-Cas9 is a complexed, two-component system using a short guide RNA (gRNA) sequence to direct the Cas9 endonuclease to the target site. Modifying the gRNA independent of the Cas9 protein confers ease and flexibility to improve the CRISPR-Cas9 system as a genome-editing tool. gRNAs have been engineered to improve the CRISPR system's overall stability, specificity, safety, and versatility. gRNAs have been modified to increase their stability to guard against nuclease degradation, thereby enhancing their efficiency. Additionally, guide specificity has been improved by limiting off-target editing. Synthetic gRNA has been shown to ameliorate inflammatory signaling caused by the CRISPR system, thereby limiting immunogenicity and toxicity in edited mammalian cells. Furthermore, through conjugation with exogenous donor DNA, engineered gRNAs have been shown to improve homology-directed repair (HDR) efficiency by ensuring donor proximity to the edited site. Lastly, synthetic gRNAs attached to fluorescent labels have been developed to enable highly specific nuclear staining and imaging, enabling mechanistic studies of chromosomal dynamics and genomic mapping. Continued work on chemical modification and optimization of synthetic gRNAs will undoubtedly lead to clinical and therapeutic benefits and, ultimately, routinely performed CRISPR-based therapies.

scholarly journals A stable DNA-free screening system for CRISPR/RNPs-mediated gene editing in hot and sweet cultivars of Capsicum annuum

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Hyeran Kim ◽  
Jisun Choi ◽  
Kang-Hee Won
Keyword(s):  
Genome Editing ◽  
Capsicum Annuum ◽  
Gene Editing ◽  
Critical Time ◽  
Sweet Pepper ◽  
Hot Pepper ◽  
Sequencing Analysis ◽  
Guide Rna ◽  
Guide Rnas

Abstract Background DNA-free, clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) ribonucleoprotein (RNP)-based genome editing is a simple, convincing, and promising tool for precision crop breeding. The efficacy of designed CRISPR-based genome editing tools is a critical prerequisite for successful precision gene editing in crops. Results This study demonstrates that soil-grown leaf- or callus-derived pepper protoplasts are a useful system for screening of efficient guide RNAs for CRISPR/Cas9 or CRISPR/Cas12a (Cpf1). CRISPR/Cas9 or Cpf1 were delivered as CRISPR/RNP complexes of purified endonucleases mixed with the designed single guide RNA, which can edit the target gene, CaMLO2 in two pepper cultivars with whole genome sequenced, Capsicum annuum ‘CM334’ and C. annuum ‘Dempsey’. The designed guide RNAs (sgRNAs for Cas9 or crRNAs for Cpf1) are conserved for CaMLO2 in both CM334 and Dempsey and cleave CaMLO2 in vitro. CRISPR/Cas9- or /Cpf1-RNP complexes were transfected into purely isolated protoplasts of the hot pepper CM334 and sweet pepper Dempsey by PEG-mediated delivery. Targeted deep sequencing analysis indicated that the targeted CaMLO2 gene was differentially edited in both cultivars, depending on the applied CRISPR/RNPs. Conclusions Pepper protoplast-based CRISPR guide-RNA selection is a robust method to check the efficacy of designed CRISPR tools and is a prerequisite for regenerating edited plants, which is a critical time-limiting procedure. The rapid and convincing selection of guide RNA against a target genome reduces the laborious efforts for tissue culture and facilitates effective gene editing for pepper improvement.

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