scholarly journals Evaluating the cleavage efficacy of CRISPR-Cas9 sgRNAs targeting ineffective regions of Arabidopsis thaliana genome

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e11409
Author(s):  
Afsheen Malik ◽  
Alvina Gul ◽  
Faiza Munir ◽  
Rabia Amir ◽  
Hadi Alipour ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Genome Editing ◽  
Statistical Tests ◽  
Gc Content ◽  
Proximal Region ◽  
Seed Region ◽  
Stem Loop ◽  
Guide Rnas ◽  
Sgrna Design ◽  
Almost All

The CRISPR-Cas9 system has recently evolved as a powerful mutagenic tool for targeted genome editing. The impeccable functioning of the system depends on the optimal design of single guide RNAs (sgRNAs) that mainly involves sgRNA specificity and on-target cleavage efficacy. Several research groups have designed algorithms and models, trained on mammalian genomes, for predicting sgRNAs cleavage efficacy. These models are also implemented in most plant sgRNA design tools due to the lack of on-target cleavage efficacy studies in plants. However, one of the major drawbacks is that almost all of these models are biased for considering only coding regions of the DNA while excluding ineffective regions, which are of immense importance in functional genomics studies especially for plants, thus making prediction less reliable. In the present study, we evaluate the on-target cleavage efficacy of experimentally validated sgRNAs designed against diverse ineffective regions of Arabidopsis thaliana genome using various statistical tests. We show that nucleotide preference in protospacer adjacent motif (PAM) proximal region, GC content in the PAM proximal seed region, intact RAR and 3rd stem loop structures, and free accessibility of nucleotides in seed and tracrRNA regions of sgRNAs are important determinants associated with their high on-target cleavage efficacy. Thus, our study describes the features important for plant sgRNAs high on-target cleavage efficacy against ineffective genomic regions previously shown to give rise to ineffective sgRNAs. Moreover, it suggests the need of developing an elaborative plant-specific sgRNA design model considering the entire genomic landscape including ineffective regions for enabling highly efficient genome editing without wasting time and experimental resources.

scholarly journals Evaluating the Efficiency of gRNAs in CRISPR/Cas9 Mediated Genome Editing in Poplars

2019 ◽  
Vol 20 (15) ◽  
pp. 3623 ◽  
Author(s):  
Tobias Bruegmann ◽  
Khira Deecke ◽  
Matthias Fladung
Keyword(s):  
Genome Editing ◽  
Gene Editing ◽  
Constitutive Expression ◽  
Gc Content ◽  
Seed Region ◽  
Research Topics ◽  
Target Region ◽  
Cas9 Nuclease ◽  
Different Types ◽  
Poplar Hybrid

CRISPR/Cas9 has become one of the most promising techniques for genome editing in plants and works very well in poplars with an Agrobacterium-mediated transformation system. We selected twelve genes, including SOC1, FUL, and their paralogous genes, four NFP-like genes and TOZ19 for three different research topics. The gRNAs were designed for editing, and, together with a constitutively expressed Cas9 nuclease, transferred either into the poplar hybrid Populus × canescens or into P. tremula. The regenerated lines showed different types of editing and revealed several homozygous editing events which are of special interest in perennial species because of limited back-cross ability. Through a time series, we could show that despite the constitutive expression of the Cas9 nuclease, no secondary editing of the target region occurred. Thus, constitutive Cas9 expression does not seem to pose any risk to additional editing events. Based on various criteria, we obtained evidence for a relationship between the structure of gRNA and the efficiency of gene editing. In particular, the GC content, purine residues in the gRNA end, and the free accessibility of the seed region seemed to be highly important for genome editing in poplars. Based on our findings on nine different poplar genes, efficient gRNAs can be designed for future efficient editing applications in poplars.

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 Integrated design, execution, and analysis of arrayed and pooled CRISPR genome editing experiments

2017 ◽  
Author(s):  
Matthew C. Canver ◽  
Maximilian Haeussler ◽  
Daniel E. Bauer ◽  
Stuart H. Orkin ◽  
Neville E. Sanjana ◽  
...  
Keyword(s):  
Genome Editing ◽  
High Throughput Sequencing ◽  
Integrated Design ◽  
Sequencing Data ◽  
Computational Tools ◽  
Guide Rnas ◽  
High Throughput Sequencing Data ◽  
Experimental Implementation ◽  
Computational Data ◽  
Sgrna Design

ABSTRACTCRISPR genome editing experiments offer enormous potential for the evaluation of genomic loci using arrayed single guide RNAs (sgRNAs) or pooled sgRNA libraries. Numerous computational tools are available to help design sgRNAs with optimal on-target efficiency and minimal off-target potential. In addition, computational tools have been developed to analyze deep sequencing data resulting from genome editing experiments. However, these tools are typically developed in isolation and oftentimes not readily translatable into laboratory-based experiments. Here we present a protocol that describes in detail both the computational and benchtop implementation of an arrayed and/or pooled CRISPR genome editing experiment. This protocol provides instructions for sgRNA design with CRISPOR, experimental implementation, and analysis of the resulting high-throughput sequencing data with CRISPResso. This protocol allows for design and execution of arrayed and pooled CRISPR experiments in 4-5 weeks by non-experts as well as computational data analysis in 1-2 days that can be performed by both computational and non-computational biologists alike.

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 Recent Advancement and Innovations in CRISPR/Cas and CRISPR Related Technologies: A review

2021 ◽  
Vol 2 (5) ◽  
pp. 01-12
Author(s):  
Sikandar Khan
Keyword(s):  
Genome Editing ◽  
Membrane Vesicles ◽  
Nano Particles ◽  
Viral Gene ◽  
Delivery Methods ◽  
Guide Rnas ◽  
Targeted Nucleases ◽  
Almost All ◽  
Technological Improvements ◽  
Viral Gene Delivery

CRISPR genome editing technologies have been improving by every passing day. The initial CRISPR/Cas9 technologies, though emerged an improved version of genome editing in competition with TALENS and ZFNs, was nevertheless not free from technical and off-target effects. Technological improvements overtime start addressing issues with original CRISPR/Cas9 technology. The major areas of improvement targeted nucleases and delivery methods. Overtime the nuclease like Cas9 had some modifications like FokI-dCas9, Truncated guide RNAs (tru-gRNAs), Paired Cas9 nickase, Cpf1, Cas6 with Csm/Csr complex and chemically treated Cas9. In terms of delivery methods the improvements came along after almost all methods including viral methods like Recombinant Adeno Associated Viruses (rAAV), Lentivirus (LV), and bacteriophages. The review summarizes various non-viral gene delivery modes including physical methods like electroporation and chemical methods like nano particles, cell-derived membrane vesicles (CMVs) with upgraded developments. The review also compares various modes of delivering CRISPR gene editing machinery.

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.

Practical R for biologists: an introduction

2021 ◽  
Keyword(s):  
Statistical Tests ◽  
Statistical Modelling ◽  
Biological Data ◽  
Early Years ◽  
Main Text ◽  
Biological Data Analysis ◽  
Base Functions ◽  
Almost All ◽  
Selection Of ◽  
R Functions

Abstract R is an open-source statistical environment modelled after the previously widely used commercial programs S and S-Plus, but in addition to powerful statistical analysis tools, it also provides powerful graphics outputs. In addition to its statistical and graphical capabilities, R is a programming language suitable for medium-sized projects. This book presents a set of studies that collectively represent almost all the R operations that beginners, analysing their own data up to perhaps the early years of doing a PhD, need. Although the chapters are organized around topics such as graphing, classical statistical tests, statistical modelling, mapping and text parsing, examples have been chosen based largely on real scientific studies at the appropriate level and within each the use of more R functions is nearly always covered than are simply necessary just to get a p-value or a graph. R comes with around a thousand base functions which are automatically installed when R is downloaded. This book covers the use of those of most relevance to biological data analysis, modelling and graphics. Throughout each chapter, the functions introduced and used in that chapter are summarized in Tool Boxes. The book also shows the user how to adapt and write their own code and functions. A selection of base functions relevant to graphics that are not necessarily covered in the main text are described in Appendix 1, and additional housekeeping functions in Appendix 2.

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