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 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 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 Multiple, Distant Gata2 Enhancers Specify Temporally and Tissue-Specific Patterning in the Developing Urogenital System

2004 ◽  
Vol 24 (23) ◽  
pp. 10263-10276 ◽  
Author(s):  
Melin Khandekar ◽  
Norio Suzuki ◽  
Jon Lewton ◽  
Masayuki Yamamoto ◽  
James Douglas Engel
Keyword(s):  
Yeast Artificial Chromosome ◽  
Artificial Chromosome ◽  
Specific Pattern ◽  
Regulatory Sequence ◽  
General Strategy ◽  
Urogenital System ◽  
Loss Of Function ◽  
Enhancer Activity ◽  
Tissue Specific ◽  
Null Mutant Mice

ABSTRACT Transcription factor GATA-2 is expressed in a complex temporally and tissue-specific pattern within the developing embryo. Loss-of-function studies in the mouse showed that GATA-2 activity is first required during very early hematopoiesis. We subsequently showed that a 271-kbp yeast artificial chromosome (YAC) transgene could fully complement the loss of Gata2 hematopoietic function but that these YAC-rescued Gata2 null mutant mice die perinatally due to defective urogenital development. The rescuing YAC did not display appropriate urogenital expression of Gata2, implying the existence of a urogenital-specific enhancer(s) lying outside the boundaries of this transgene. Here we outline a coupled general strategy for regulatory sequence discovery, linking bioinformatics to functional genomics based on the bacterial artificial chromosome (BAC) libraries used to generate the mouse genome sequence. Exploiting this strategy, we screened >1 Mbp of genomic DNA surrounding Gata2 for urogenital enhancer activity. We found that the spatially and tissue-specific functions for Gata2 in the developing urogenital system are conferred by at least three separate regionally and temporally specific urogenital enhancer elements, two of which reside far 3′ to the Gata2 structural gene. Including the additional enhancers that were discovered using this strategy (called BAC trap) extends the functional realm of the Gata2 locus to greater than 1 Mbp.

scholarly journals Vaccinia Virus WR Gene A5L Is Required for Morphogenesis of Mature Virions

1999 ◽  
Vol 73 (6) ◽  
pp. 4590-4599 ◽  
Author(s):  
Ollie Williams ◽  
Elizabeth J. Wolffe ◽  
Andrea S. Weisberg ◽  
Michael Merchlinsky
Keyword(s):  
Vaccinia Virus ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Endogenous Gene ◽  
Late Protein ◽  
Full Complement ◽  
Immature Virion ◽  
Infected Cells ◽  
Synthesis And Processing ◽  
Vaccinia Virion

ABSTRACT The vaccinia virus WR A5L open reading frame (corresponding to open reading frame A4L in vaccinia virus Copenhagen) encodes an immunodominant late protein found in the core of the vaccinia virion. To investigate the role of this protein in vaccinia virus replication, we have constructed a recombinant virus, vA5Li, in which the endogenous gene has been deleted and an inducible copy of the A5 gene dependent on isopropyl-β-d-thiogalactopyranoside (IPTG) for expression has been inserted into the genome. In the absence of inducer, the yield of infectious virus was dramatically reduced. However, DNA synthesis and processing, viral protein expression (except for A5), and early stages in virion formation were indistinguishable from the analogous steps in a normal infection. Electron microscopy revealed that the major vaccinia virus structural form present in cells infected with vA5Li in the absence of inducer was immature virions. Viral particles were purified from vA5Li-infected cells in the presence and absence of inducer. Both particles contained viral DNA and the full complement of viral proteins, except for A5, which was missing from particles prepared in the absence of inducer. The particles prepared in the presence of IPTG were more infectious than those prepared in its absence. The A5 protein appears to be required for the immature virion to form the brick-shaped intracellular mature virion.

scholarly journals Tissue-specific dynamic codon redefinition in Drosophila

2020 ◽  
Author(s):  
Andrew M. Hudson ◽  
Gary Loughran ◽  
Nicholas L. Szabo ◽  
Norma M. Wills ◽  
John F. Atkins ◽  
...  
Keyword(s):  
High Efficiency ◽  
Stop Codon ◽  
Open Reading Frame ◽  
Stem Loop ◽  
Tissue Specific ◽  
Reading Frame ◽  
Cis Acting ◽  
A Subunit ◽  
Specific Regulation ◽  
Stop Codon Readthrough

Stop codon readthrough during translation occurs in many eukaryotes, including Drosophila, yeast, and humans. Recoding of UGA, UAG or UAA to specify an amino acid allows the ribosome to synthesize C-terminally extended proteins. We previously found evidence for tissue-specific regulation of stop codon readthrough in decoding the Drosophila kelch gene, whose first open reading frame (ORF1) encodes a subunit of a Cullin3-RING ubiquitin ligase. Here, we show that the efficiency of kelch readthrough varies markedly by tissue. Immunoblotting for Kelch ORF1 protein revealed high levels of the readthrough product in lysates of larval and adult central nervous system (CNS) tissue and larval imaginal discs. A sensitive reporter of kelch readthrough inserted after the second kelch open reading frame (ORF2) directly detected synthesis of Kelch readthrough product in these tissues. To analyze the role of cis-acting sequences in regulating kelch readthrough, we used cDNA reporters to measure readthrough in both transfected human cells and transgenic Drosophila. Results from a truncation series suggest that a predicted mRNA stem-loop 3’ of the ORF1 stop codon stimulates high-efficiency readthrough. Expression of cDNA reporters using cell type-specific Gal4 drivers revealed that CNS readthrough is restricted to neurons. Finally, we show that high-effficiency readthrough in the CNS is common in Drosophila, raising the possibility that the neuronal proteome includes many proteins with conserved C-terminal extensions. This work provides new evidence for a remarkable degree of tissue- and cell-specific dynamic stop codon redefinition in Drosophila.

Cloning and analysis of unique human glutaminase isoforms generated by tissue-specific alternative splicing

1999 ◽  
Vol 1 (2) ◽  
pp. 51-62 ◽  
Author(s):  
KHALED M. ELGADI ◽  
ROBERT A. MEGUID ◽  
MING QIAN ◽  
WILEY W. SOUBA ◽  
STEVE F. ABCOUWER
Keyword(s):  
Alternative Splicing ◽  
Rat Kidney ◽  
Cancer Cell Line ◽  
Open Reading Frame ◽  
High Rate ◽  
Computer Search ◽  
Tissue Specific ◽  
Reading Frame ◽  
Mrna Species ◽  
Specific Alternative

Elgadi, Khaled M., Robert A. Meguid, Ming Qian, Wiley W. Souba, and Steve F. Abcouwer. Cloning and analysis of unique human glutaminase isoforms generated by tissue-specific alternative splicing. Physiol. Genomics 1: 51–62, 1999.—Three human glutaminase (hGA) isoforms were identified, two of which represent isoforms previously unidentified in any species. One isoform contains an open reading frame with high homology with the rat kidney-type glutaminase, suggesting that this isoform represents the human kidney-type glutaminase, hKGA. A second isoform, termed hGAC, contains an open reading frame that matches hKGA except for a unique COOH-terminal amino acid sequence. In addition, a third human glutaminase isoform was identified from a computer search and on further analysis was found to represent an additional unique isoform, hGAM. hKGA is expressed predominantly in brain and kidney but not in liver, hGAC is expressed principally in cardiac muscle and pancreas but not in liver or brain, and hGAM is expressed solely in cardiac and skeletal muscle. hGAC is the predominant isoform expressed by a human breast cancer cell line that exhibits a high rate of glutamine utilization and glutaminase activity. Genomic Southern analysis as well as isolation and analysis of five glutaminase genomic clones suggested that all three hGA isoforms originate from the same locus and therefore represent mRNA species that are produced by tissue-specific alternative splicing of a single pre-mRNA. Furthermore, an RT-PCR assay was developed that can be used to easily differentiate between hKGA and hGAC mRNA species.

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.

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