scholarly journals Proxies of CRISPR/Cas9 Activity To Aid in the Identification of Mutagenized Arabidopsis Plants

2020 ◽  
Vol 10 (6) ◽  
pp. 2033-2042 ◽  
Author(s):  
Renyu Li ◽  
Charles Vavrik ◽  
Cristian H. Danna
Keyword(s):  
Arabidopsis Thaliana ◽  
Gene Function ◽  
Good Predictor ◽  
Gene Editing ◽  
Selection Strategy ◽  
Directed Mutagenesis ◽  
Loss Of Function ◽  
Editing Event ◽  
Endogenous Gene ◽  
Non Coding Rnas

CRISPR/Cas9 has become the preferred gene-editing technology to obtain loss-of-function mutants in plants, and hence a valuable tool to study gene function. This is mainly due to the easy reprogramming of Cas9 specificity using customizable small non-coding RNAs, and to the possibility of editing several independent genes simultaneously. Despite these advances, the identification of CRISPR-edited plants remains time and resource-intensive. Here, based on the premise that one editing event in one locus is a good predictor of editing event/s in other locus/loci, we developed a CRISPR co-editing selection strategy that greatly facilitates the identification of CRISPR-mutagenized Arabidopsis thaliana plants. This strategy is based on targeting the gene/s of interest simultaneously with a proxy of CRISPR-Cas9-directed mutagenesis. The proxy is an endogenous gene whose loss-of-function produces an easy-to-detect visible phenotype that is unrelated to the expected phenotype of the gene/s under study. We tested this strategy via assessing the frequency of co-editing of three functionally unrelated proxy genes. We found that each proxy predicted the occurrence of mutations in each surrogate gene with efficiencies ranging from 68 to 100%. The selection strategy laid out here provides a framework to facilitate the identification of multiplex edited plants, thus aiding in the study of gene function when functional redundancy hinders the effort to define gene-function-phenotype links.

scholarly journals Proxies of CRISPR-Cas9 activity to aid in the identification of mutagenized Arabidopsis plants

2019 ◽  
Author(s):  
Renyu Li ◽  
Charles Vavrik ◽  
Cristian H. Danna
Keyword(s):  
Gene Function ◽  
Gene Editing ◽  
Gene Families ◽  
Single Copy ◽  
Selection Strategy ◽  
Loss Of Function ◽  
Editing Event ◽  
Endogenous Gene ◽  
Polyploid Plant ◽  
Non Coding Rnas

AbstractCRISPR-Cas9 has become the preferred gene editing technology to obtain loss-of-function mutants in plants, and hence a valuable tool to study gene function. This is mainly due to the easy reprograming of Cas9 specificity using customizable small non-coding RNAs, and to the ability to target several independent genes simultaneously. Despite these advances, the identification of CRISPR-edited plants remains time and resource consuming. Here, based on the premise that one editing event in one locus is a good predictor of editing event/s in other locus/loci, we developed a CRISPR co-editing selection strategy that greatly facilitates the identification of CRISPR-mutagenized Arabidopsis plants. This strategy is based on targeting the gene/s of interest simultaneously with a proxy of CRISPR-Cas9-directed mutagenesis. The proxy is an endogenous gene whose loss-of-function mutation produces an easy-to-detect visible phenotype that is unrelated to the expected phenotype of the gene/s under study. We tested this strategy via assessing the frequency of co-editing of three functionally unrelated proxies. We found all three proxies predicted the occurrence of mutations in either or both of the other two proxies with efficiencies ranging from 40% to 100%, dramatically reducing the number of plants that need to be screened to identify CRISPR mutants. This selection strategy provides a framework to facilitate gene function studies of gene families as well as the function of single copy genes in polyploid plant species where the identification of multiplex mutants remains challenging.

scholarly journals A gene-editing/complementation strategy for tissue-specific lignin reduction while preserving biomass yield

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Hasi Yu ◽  
Chang Liu ◽  
Richard A. Dixon
Keyword(s):  
Gene Editing ◽  
Biomass Yield ◽  
Lignin Content ◽  
Open Reading Frame ◽  
General Strategy ◽  
Loss Of Function ◽  
Tissue Specific ◽  
Knock Out ◽  
Reading Frame ◽  
Endogenous Gene

Abstract Background Lignification of secondary cell walls is a major factor conferring recalcitrance of lignocellulosic biomass to deconstruction for fuels and chemicals. Genetic modification can reduce lignin content and enhance saccharification efficiency, but usually at the cost of moderate-to-severe growth penalties. We have developed a method, using a single DNA construct that uses CRISPR–Cas9 gene editing to knock-out expression of an endogenous gene of lignin monomer biosynthesis while at the same time expressing a modified version of the gene’s open reading frame that escapes cutting by the Cas9 system and complements the introduced mutation in a tissue-specific manner. Results Expressing the complementing open reading frame in vessels allows for the regeneration of Arabidopsis plants with reduced lignin, wild-type biomass yield, and up to fourfold enhancement of cell wall sugar yield per plant. The above phenotypes are seen in both homozygous and bi-allelic heterozygous T1 lines, and are stable over at least four generations. Conclusions The method provides a rapid approach for generating reduced lignin trees or crops with one single transformation event, and, paired with a range of tissue-specific promoters, provides a general strategy for optimizing loss-of-function traits that are associated with growth penalties. This method should be applicable to any plant species in which transformation and gene editing are feasible and validated vessel-specific promoters are available.

scholarly journals Highly efficient CRISPR gene editing in yeast enabled by double selection

2018 ◽  
Author(s):  
Philippe C Després ◽  
Alexandre K Dubé ◽  
Lou Nielly-Thibault ◽  
Nozomu Yachie ◽  
Christian R Landry
Keyword(s):  
High Frequency ◽  
Large Scale ◽  
Gene Editing ◽  
Chromosomal Translocations ◽  
Selection Strategy ◽  
Loss Of Function ◽  
Base Editing ◽  
Fitness Effects ◽  
Genetics And Genomics ◽  
Selection For

AbstractCRISPR-Cas9 loss of function (LOF) and base editing screens are powerful tools in genetics and genomics. Yeast is one of the main models in genetics and genomics, yet large-scale approaches remain to be developed in this species because of low mutagenesis rates without donor DNA. We developed a double selection strategy based on co-selection that increases LOF mutation rates, both for CRISPR-Cas9 and the Target-AID base editor. We constructed the pDYSCKO vector, which is amenable to high throughput double selection for both approaches. Using modeling, we show that this improvement provides the required increased in detection power to measure the fitness effects of thousands of mutations in typical yeast pooled screens. We also show that multiplex genome editing with Cas9 causes programmable chromosomal translocations at high frequency, suggesting that multiplex editing should be performed with caution and that base-editors could be preferable tools for LOF screens.

scholarly journals A dual sgRNA approach for functional genomics in Arabidopsis thaliana[OPEN]

2017 ◽  
Author(s):  
Laurens Pauwels ◽  
Rebecca De Clercq ◽  
Jonas Goossens ◽  
Sabrina Iñigo ◽  
Clara Williams ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Gene Editing ◽  
Reverse Genetics ◽  
Gene Families ◽  
Null Alleles ◽  
Loss Of Function ◽  
Knock Out ◽  
Nonsense Mediated Decay ◽  
Dna Insertion ◽  
Entire Gene

AbstractReverse genetics uses loss-of-function alleles to interrogate gene function. The advent of CRISPR/Cas9-based gene editing now allows to generate knock-out alleles for any gene and entire gene families. Even in the model plant Arabidopsis thaliana, gene editing is welcomed as T-DNA insertion lines do not always generate null alleles. Here, we show efficient generation of heritable mutations in Arabidopsis using CRISPR/Cas9 with a workload similar to generating overexpression lines. We obtain Cas9 null-segregants with bi-allelic mutations in the T2 generation. Out of seven new mutant alleles we report here, one allele for GRXS17, the ortholog of human GRX3/PICOT, did not phenocopy previously characterized nulls. Notwithstanding, the mutation caused a frameshift and triggered nonsense-mediated decay. We demonstrate that our workflow is also compatible with a dual sgRNA approach in which a gene is targeted by two sgRNAs simultaneously. This paired nuclease method can result in a more reliable loss-of-function alleles that lack a large essential part of the gene. The ease in the CRISPR/Cas9 workflow should help in the eventual generation of true null alleles of every gene in the Arabidopsis genome, which will advance both basic and applied plant research.One-sentence summaryWe present a dual sgRNA approach to delete Arabidopsis gene 34 fragments in order to obtain reliable functional knock-outs.

scholarly journals Bar-seq strategies for the LeishGEdit toolbox

2020 ◽  
Author(s):  
Tom Beneke ◽  
Eva Gluenz
Keyword(s):  
High Throughput ◽  
Gene Function ◽  
Gene Editing ◽  
Primer Design ◽  
List Type ◽  
Loss Of Function ◽  
Link Type ◽  
Analysis Tools ◽  
Mixed Populations

AbstractThe number of fully sequenced genomes increases steadily but the function of many genes remains unstudied. To accelerate dissection of gene function in Leishmania spp. and other kinetoplastids we developed previously a streamlined pipeline for CRISPR-Cas9 gene editing, which we termed LeishGEdit [1]. To facilitate high-throughput mutant screens we have adapted this pipeline by barcoding mutants with unique 17-nucleotide barcodes, allowing loss-of-function screens in mixed populations [2]. Here we present primer design and analysis tools that facilitate these bar-seq strategies. We have developed a standalone easy-to-use pipeline to design CRISPR primers suitable for the LeishGEdit toolbox for any given genome and have generated a list of 14,995 barcodes. Barcodes and oligos are now accessible through our website www.leishgedit.net allowing to pursue bar-seq experiments in all currently available TriTrypDB genomes (release 41). This will streamline CRISPR bar-seq assays in kinetoplastids, enabling pooled mutant screens across the community.HighlightsDeveloping tools for pooled bar-seq mutant screens across the kinetoplastid communityDevelopment of a standalone script to design primers suitable for the LeishGEdit toolboxGeneration of 14,995 barcodes that can be used for bar-seq strategies in kinetoplastidsBar-seq primers for all TriTrypDB genomes (release 41) can be obtained from www.leishgedit.net

scholarly journals Latest Advances of Virology Research Using CRISPR/Cas9-Based Gene-Editing Technology and Its Application to Vaccine Development

Viruses ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 779
Author(s):  
Man Teng ◽  
Yongxiu Yao ◽  
Venugopal Nair ◽  
Jun Luo
Keyword(s):  
Biological Sciences ◽  
Gene Therapy ◽  
Genetic Engineering ◽  
Gene Function ◽  
Viral Genome ◽  
Gene Editing ◽  
Vaccine Development ◽  
Future Prospects ◽  
Dna Viruses ◽  
Viral Genomes

In recent years, the CRISPR/Cas9-based gene-editing techniques have been well developed and applied widely in several aspects of research in the biological sciences, in many species, including humans, animals, plants, and even in viruses. Modification of the viral genome is crucial for revealing gene function, virus pathogenesis, gene therapy, genetic engineering, and vaccine development. Herein, we have provided a brief review of the different technologies for the modification of the viral genomes. Particularly, we have focused on the recently developed CRISPR/Cas9-based gene-editing system, detailing its origin, functional principles, and touching on its latest achievements in virology research and applications in vaccine development, especially in large DNA viruses of humans and animals. Future prospects of CRISPR/Cas9-based gene-editing technology in virology research, including the potential shortcomings, are also discussed.

scholarly journals The efficacy of CRISPR-mediated cytosine base editing with the RPS5a promoter in Arabidopsis thaliana

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Minkyung Choi ◽  
Jae-Young Yun ◽  
Jun-Hyuk Kim ◽  
Jin-Soo Kim ◽  
Sang-Tae Kim
Keyword(s):  
Arabidopsis Thaliana ◽  
Cytidine Deaminase ◽  
Biological Research ◽  
Loss Of Function ◽  
Nucleotide Substitutions ◽  
Viral Origin ◽  
Nonsense Mutations ◽  
Base Editing ◽  
Low Efficiency ◽  
Viable System

AbstractCRISPR/Cas9-mediated genome editing is an important and versatile technology in modern biological research. Recent advancements include base-editing CRISPR tools that enable targeted nucleotide substitutions using a fusion protein comprising a nickase variant of Cas9 and a base deaminase. Improvements in base editing efficiencies and inheritable of edited loci need to be made to make CRISPR a viable system in plants. Here, we report efficiency of cytosine base editors (CBEs) in Arabidopsis thaliana by applying the strong endogenous RPS5a promoter to drive the expression of nickase Cas9 and either rAPOBEC1 from rat (BE3) or the PmCDA1 activation-induced cytidine deaminase from sea lamprey (AIDv2). Compared with the strong heterologous CaMV35S promoter of viral origin, the RPS5a promoter improved CBE efficiency by 32% points with the number of T1 plants showing over 50% conversion ratio when the LFY gene was targeted. CBE induced nonsense mutations in LFY via C-to-T conversion, which resulted in loss-of-function lfy phenotypes; defects in LFY function were associated with the targeted base substitutions. Our data suggest that optimal promoter choice for CBE expression may affect base-editing efficiencies in plants. The results provide a strategy to optimize low-efficiency base editors and demonstrate their applicability for functional assays and trait development in crop research.

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