scholarly journals CRISPR/Cas9 with single guide RNA expression driven by small tRNA promoters showed reduced editing efficiency compared to a U6 promoter

RNA ◽  
2016 ◽  
Vol 23 (1) ◽  
pp. 1-5 ◽  
Author(s):  
Yuda Wei ◽  
Yan Qiu ◽  
Yanhao Chen ◽  
Gaigai Liu ◽  
Yongxian Zhang ◽  
...  
Keyword(s):  
Rna Expression ◽  
Editing Efficiency ◽  
Guide Rna ◽  
U6 Promoter

scholarly journals A multiplex guide RNA expression system and its efficacy for plant genome engineering

2020 ◽  
Author(s):  
Youngbin Oh ◽  
Hyeonjin Kim ◽  
Bora Lee ◽  
Sang-Gyu Kim
Keyword(s):  
Genome Engineering ◽  
Binary Vector ◽  
Expression System ◽  
Plant Genome ◽  
Nicotiana Attenuata ◽  
Specific Sequence ◽  
Editing Efficiency ◽  
Guide Rna ◽  
Assembly Method ◽  
A Genome

Abstract BackgroundThe Streptococcus pyogenes CRISPR system is composed of a Cas9 endonuclease (SpCas9) and a single-stranded guide RNA (gRNA) harboring a target-specific sequence. Theoretically, SpCas9 proteins could cleave as many targeted loci as gRNAs bind in a genome.ResultsWe introduce a PCR-free multiple gRNA cloning system for editing plant genomes. This method consists of two steps: (1) cloning annealed products of two oligonucleotides harboring target-binding sequence between tRNA and gRNA scaffold sequences in a pGRNA vector; and (2) assembling tRNA-gRNA units from several pGRNA vectors with a plant binary vector containing a SpCas9 expression cassette using the Golden Gate assembly method. We validated the editing efficiency and patterns of the multiplex gRNA expression system in wild tobacco (Nicotiana attenuata) protoplasts and in transformed plants by performing targeted deep sequencing. Two proximal cleavages by SpCas9-gRNA largely increased the editing efficiency and induced large deletions between two cleavage sites.ConclusionsThis multiplex gRNA expression system enables high-throughput production of a single binary vector and increases the efficiency of plant genome editing.

scholarly journals Programmable RNA base editing with a single gRNA-free enzyme

2021 ◽  
Author(s):  
Wenjian Han ◽  
Wendi Huang ◽  
Miaowei Mao ◽  
Tong Wei ◽  
Yanwen Ye ◽  
...  
Keyword(s):  
Rna Editing ◽  
Rna Binding ◽  
Artificial Enzyme ◽  
Free System ◽  
Editing Efficiency ◽  
Base Editing ◽  
Guide Rna ◽  
Human Proteins ◽  
Target Effect ◽  
Puf Domain

ABSTRACTProgrammable RNA editing enables rewriting gene expression without changing genome sequences. Current tools for specific RNA editing dependent on the assembly of guide RNA into an RNA/protein complex, causing delivery barrier and low editing efficiency. We report a new gRNA-free system, RNA editing with individual RNA-binding enzyme (REWIRE), to perform precise base editing with a single engineered protein. This artificial enzyme contains a human-originated programmable PUF domain to specifically recognize RNAs and different deaminase domains to achieve efficient A-to-I or C-to-U editing, which achieved 60-80% editing rate in human cells, with a few non-specific editing sites in the targeted region and a low level off-target effect globally. The RNA-binding domain in REWIREs was further optimized to improve editing efficiency and minimize off-target effects. We applied the REWIREs to correct disease-associated mutations and achieve both types of base editing in mice. As a single-component system originated from human proteins, REWIRE presents a precise and efficient RNA editing platform with broad applicability.

scholarly journals Decoupling tRNA promoter and processing activities enables specific Pol-II Cas9 guide RNA expression

2018 ◽  
Author(s):  
David JHF Knapp ◽  
Yale S Michaels ◽  
Max Jamilly ◽  
Quentin RV Ferry ◽  
Hector Barbosa ◽  
...  
Keyword(s):  
Rational Design ◽  
Rna Expression ◽  
Temporal Control ◽  
High Background ◽  
Pol Ii ◽  
Trna Processing ◽  
Guide Rna ◽  
Promising Strategy ◽  
Screening Platform

ABSTRACTSpatial/temporal control of Cas9 guide RNA expression could considerably expand the utility of CRISPR-based technologies. Current approaches based on tRNA processing offer a promising strategy but suffer from high background. Here we developed a variant screening platform to identify differential sequence determinants of human tRNA promoter and processing activities. Rational design based on the ensuing principles allowed us to engineer an improved tRNA scaffold that enabled highly specific guide RNA production from a Pol-II promoter.

scholarly journals Highly multiplex guide RNA expression units of CRISPR/Cas9 were completely stable using cosmid amplification in a novel polygonal structure

2019 ◽  
Vol 21 (11) ◽  
Author(s):  
Tomoko Nakanishi ◽  
Aya Maekawa ◽  
Hirotaka Tabata ◽  
Takashi Yoshioka ◽  
Zheng Pei ◽  
...  
Keyword(s):  
Rna Expression ◽  
Guide Rna ◽  
Polygonal Structure

Optimisation of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 : single-guide RNA (sgRNA) delivery system in a goat model

2019 ◽  
Vol 31 (9) ◽  
pp. 1533 ◽  
Author(s):  
Yu Huang ◽  
Yige Ding ◽  
Yao Liu ◽  
Shiwei Zhou ◽  
Qiang Ding ◽  
...  
Keyword(s):  
Delivery System ◽  
Large Animal ◽  
Editing Efficiency ◽  
Guide Rna ◽  
Targeting Efficiency ◽  
Sgrna Design ◽  
Large Animals ◽  
Animal Genomes ◽  
Selection Of ◽  
Cas9 Mrna

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is an efficient method for the production of gene-edited animals. We have successfully generated gene-modified goats and sheep via zygote injection of Cas9 mRNA and single-guide RNA (sgRNA) mixtures. However, the delivery system for microinjection largely refers to methods established for mice; optimised injection conditions are urgently required for the generation of large animals. Here, we designed a study to optimise the Cas9 mRNA and sgRNA delivery system for goats. By comparing four computational tools for sgRNA design and validating the targeting efficiency in goat fibroblasts, we suggest a protocol for the selection of desirable sgRNAs with higher targeting efficiency and negligible off-target mutations. We further evaluated the editing efficiency in goat zygotes injected with Cas9:sgRNA (sg8) and found that injection with 50ngμL−1 Cas9 mRNA and 25ngμL−1 sgRNA yielded an increased editing efficiency. Our results provide a reference protocol for the optimisation of the injection conditions for the efficient editing of large animal genomes via the zygote injection approach.

scholarly journals A multiplex guide RNA expression system and its efficacy for plant genome engineering

Plant Methods ◽  
2020 ◽  
Vol 16 (1) ◽  
Author(s):  
Youngbin Oh ◽  
Bora Lee ◽  
Hyeonjin Kim ◽  
Sang-Gyu Kim
Keyword(s):  
Genome Engineering ◽  
Expression System ◽  
Plant Genome ◽  
Rna Expression ◽  
Guide Rna

scholarly journals A multiplex guide RNA expression system and its efficacy for plant genome engineering

2020 ◽  
Author(s):  
Youngbin Oh ◽  
Bora Lee ◽  
Hyeonjin Kim ◽  
Sang-Gyu Kim
Keyword(s):  
Genome Engineering ◽  
Binary Vector ◽  
Expression System ◽  
Plant Genome ◽  
Nicotiana Attenuata ◽  
Specific Sequence ◽  
Editing Efficiency ◽  
Guide Rna ◽  
Assembly Method ◽  
A Genome

Abstract Background: The Streptococcus pyogenes CRISPR system is composed of a Cas9 endonuclease (SpCas9) and a single-stranded guide RNA (gRNA) harboring a target-specific sequence. Theoretically, SpCas9 proteins could cleave as many targeted loci as gRNAs bind in a genome.Results: We introduce a PCR-free multiple gRNA cloning system for editing plant genomes. This method consists of two steps: (1) cloning the annealed products of two single-stranded oligonucleotide fragments harboring a complimentary target-binding sequence on each strand between tRNA and gRNA scaffold sequences in a pGRNA vector; and (2) assembling tRNA-gRNA units from several pGRNA vectors with a plant binary vector containing a SpCas9 expression cassette using the Golden Gate assembly method. We validated the editing efficiency and patterns of the multiplex gRNA expression system in wild tobacco (Nicotiana attenuata) protoplasts and in transformed plants by performing targeted deep sequencing. Two proximal cleavages by SpCas9-gRNA largely increased the editing efficiency and induced large deletions between two cleavage sites.Conclusions: This multiplex gRNA expression system enables high-throughput production of a single binary vector and increases the efficiency of plant genome editing.

scholarly journals A multiplex guide RNA expression system and its efficacy for plant genome engineering

2020 ◽  
Author(s):  
Youngbin Oh ◽  
Bora Lee ◽  
Hyeonjin Kim ◽  
Sang-Gyu Kim
Keyword(s):  
Genome Engineering ◽  
Binary Vector ◽  
Expression System ◽  
Plant Genome ◽  
Nicotiana Attenuata ◽  
Specific Sequence ◽  
Editing Efficiency ◽  
Guide Rna ◽  
Assembly Method ◽  
A Genome

Abstract Background The Streptococcus pyogenes CRISPR system is composed of a Cas9 endonuclease (SpCas9) and a single-stranded guide RNA (gRNA) harboring a target-specific sequence. Theoretically, SpCas9 proteins could cleave as many targeted loci as gRNAs bind in a genome. Results We introduce a PCR-free multiple gRNA cloning system for editing plant genomes. This method consists of two steps: (1) cloning the annealed products of two single-stranded oligonucleotide fragments harboring a complimentary target-binding sequence on each strand between tRNA and gRNA scaffold sequences in a pGRNA vector; and (2) assembling tRNA-gRNA units from several pGRNA vectors with a plant binary vector containing a SpCas9 expression cassette using the Golden Gate assembly method. We validated the editing efficiency and patterns of the multiplex gRNA expression system in wild tobacco (Nicotiana attenuata) protoplasts and in transformed plants by performing targeted deep sequencing. Two proximal cleavages by SpCas9-gRNA largely increased the editing efficiency and induced large deletions between two cleavage sites. Conclusions This multiplex gRNA expression system enables high-throughput production of a single binary vector and increases the efficiency of plant genome editing.

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