Correction to: A Procedure to Design Guide RNA, Assemble Fragments, and Detect Mutation for Genome Editing in Flax

2020 ◽  
pp. C1-C1
Author(s):  
Dinesh Adhikary ◽  
Isadora Louise Alves da Costa Ribeiro Quintans ◽  
Pankaj K. Bhowmik
Keyword(s):  
Genome Editing ◽  
Guide Rna ◽  
Design Guide

A Procedure to Design Guide RNA, Assemble Fragments, and Detect Mutation for Genome Editing in Flax

2020 ◽  
pp. 173-190
Author(s):  
Dinesh Adhikary ◽  
Isadora Louise Alves da Costa Ribeiro Quintans ◽  
Pankaj K. Bhowmik
Keyword(s):  
Genome Editing ◽  
Guide Rna ◽  
Design Guide

Co-encapsulation of Cas9 mRNA and guide RNA in polyplex micelles enables genome editing in mouse brain

2021 ◽  
Vol 332 ◽  
pp. 260-268
Author(s):  
Saed Abbasi ◽  
Satoshi Uchida ◽  
Kazuko Toh ◽  
Theofilus A. Tockary ◽  
Anjaneyulu Dirisala ◽  
...  
Keyword(s):  
Genome Editing ◽  
Mouse Brain ◽  
Guide Rna ◽  
Cas9 Mrna

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.

scholarly journals CRISPR/Cas9-mediated targeted mutagenesis in Japanese cedar (Cryptomeria japonica D. Don)

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yoshihiko Nanasato ◽  
Masafumi Mikami ◽  
Norihiro Futamura ◽  
Masaki Endo ◽  
Mitsuru Nishiguchi ◽  
...  
Keyword(s):  
Genome Editing ◽  
Cryptomeria Japonica ◽  
Fluorescent Protein ◽  
Japanese Cedar ◽  
Targeted Mutagenesis ◽  
Embryogenic Tissue ◽  
Breeding Period ◽  
Single Mutation ◽  
Knock Out ◽  
Guide Rna

AbstractCryptomeria japonica (Japanese cedar or sugi) is one of the most important coniferous tree species in Japan and breeding programs for this species have been launched since 1950s. Genome editing technology can be used to shorten the breeding period. In this study, we performed targeted mutagenesis using the CRISPR/Cas9 system in C. japonica. First, the CRISPR/Cas9 system was tested using green fluorescent protein (GFP)-expressing transgenic embryogenic tissue lines. Knock-out efficiency of GFP ranged from 3.1 to 41.4% depending on U6 promoters and target sequences. The GFP knock-out region was mottled in many lines, indicating genome editing in individual cells. However, in 101 of 102 mutated individuals (> 99%) from 6 GFP knock-out lines, embryos had a single mutation pattern. Next, we knocked out the endogenous C. japonica magnesium chelatase subunit I (CjChlI) gene using two guide RNA targets. Green, pale green, and albino phenotypes were obtained in the gene-edited cell lines. Sequence analysis revealed random deletions, insertions, and replacements in the target region. Thus, targeted mutagenesis using the CRISPR/Cas9 system can be used to modify the C. japonica genome.

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 CRISPR-Cas9 delivery to hard-to-transfect cells via membrane deformation

2015 ◽  
Vol 1 (7) ◽  
pp. e1500454 ◽  
Author(s):  
Xin Han ◽  
Zongbin Liu ◽  
Myeong chan Jo ◽  
Kai Zhang ◽  
Ying Li ◽  
...  
Keyword(s):  
Genome Editing ◽  
Function Analysis ◽  
Embryonic Stem ◽  
Cell Types ◽  
Disease Genes ◽  
Deformation Method ◽  
Targeting Therapy ◽  
Membrane Deformation ◽  
Guide Rna ◽  
Different Cell Types

The CRISPR (clustered regularly interspaced short palindromic repeats)–Cas (CRISPR-associated) nuclease system represents an efficient tool for genome editing and gene function analysis. It consists of two components: single-guide RNA (sgRNA) and the enzyme Cas9. Typical sgRNA and Cas9 intracellular delivery techniques are limited by their reliance on cell type and exogenous materials as well as their toxic effects on cells (for example, electroporation). We introduce and optimize a microfluidic membrane deformation method to deliver sgRNA and Cas9 into different cell types and achieve successful genome editing. This approach uses rapid cell mechanical deformation to generate transient membrane holes to enable delivery of biomaterials in the medium. We achieved high delivery efficiency of different macromolecules into different cell types, including hard-to-transfect lymphoma cells and embryonic stem cells, while maintaining high cell viability. With the advantages of broad applicability across different cell types, particularly hard-to-transfect cells, and flexibility of application, this method could potentially enable new avenues of biomedical research and gene targeting therapy such as mutation correction of disease genes through combination of the CRISPR-Cas9–mediated knockin system.

scholarly journals Development of Beet necrotic yellow vein virus ‐based vectors for multiple‐gene expression and guide RNA delivery in plant genome editing

2019 ◽  
Vol 17 (7) ◽  
pp. 1302-1315 ◽  
Author(s):  
Ning Jiang ◽  
Chao Zhang ◽  
Jun‐Ying Liu ◽  
Zhi‐Hong Guo ◽  
Zong‐Ying Zhang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Genome Editing ◽  
Plant Genome ◽  
Yellow Vein ◽  
Multiple Gene ◽  
Guide Rna

scholarly journals CRISPR-Cas12a (Cpf1): A Versatile Tool in the Plant Genome Editing Tool Box for Agricultural Advancement

2020 ◽  
Author(s):  
Anindya Bandyopadhyay ◽  
Nagesh Kancharla ◽  
vivek javalkote ◽  
santanu dasgupta ◽  
Thomas Brutnell
Keyword(s):  
Genome Editing ◽  
Genome Engineering ◽  
Temperature Stability ◽  
Double Strand Breaks ◽  
Plant Genome ◽  
Strand Breaks ◽  
Guide Rna ◽  
Versatile Tool ◽  
Type V ◽  
Global Population

Global population is predicted to approach 10 billion by 2050, an increase of over 2 billion from today. To meet the demands of growing, geographically and socio-economically diversified nations, we need to diversity and expand agricultural production. This expansion of agricultural productivity will need to occur under increasing biotic, and environmental constraints driven by climate change. Clustered regularly interspaced short palindromic repeats-site directed nucleases (CRISPR-SDN) and similar genome editing technologies will likely be key enablers to meet future agricultural needs. While the application of CRISPR-Cas9 mediated genome editing has led the way, the use of CRISPR-Cas12a is also increasing significantly for genome engineering of plants. The popularity of the CRISPR-Cas12a, the type V (class-II) system, is gaining momentum because of its versatility and simplified features. These include the use of a small guide RNA devoid of trans-activating crispr RNA (tracrRNA), targeting of T-rich regions of the genome where Cas9 is not suitable for use, RNA processing capability facilitating simpler multiplexing, and its ability to generate double strand breaks (DSB) with staggered ends. Many monocot and dicot species have been successfully edited using this Cas12a system and further research is ongoing to improve its efficiency in plants, including improving the temperature stability of the Cas12a enzyme, identifying new variants of Cas12a or synthetically producing Cas12a with flexible PAM sequences. In this review we provide a comparative survey of CRISPR-Cas12a and Cas9, and provide a perspective on applications of CRISPR-Cas12 in agriculture.

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 CRISPR Explorer: A fast and intuitive tool for designing guide RNA for genome editing

2016 ◽  
Vol 3 (4) ◽  
pp. 56 ◽  
Author(s):  
Kenian Chen ◽  
Yi Jin ◽  
Yin C. Lin
Keyword(s):  
Genome Editing ◽  
Guide Rna
Sign in / Sign up

Export Citation Format

Share Document