scholarly journals CRISPys: Optimal sgRNA design for editing multiple members of a gene family using the CRISPR system

2017 ◽  
Author(s):  
Gal Hyams ◽  
Shiran Abadi ◽  
Adi Avni ◽  
Eran Halperin ◽  
Eilon Shani ◽  
...  
Keyword(s):  
Optimal Design ◽  
Gene Family ◽  
Genome Editing ◽  
Reverse Genetics ◽  
Gene Knockout ◽  
Gene Families ◽  
The Other ◽  
List Type ◽  
Suggested Approach ◽  
Eukaryotic Genomes

AbstractThe discovery and development of the CRISPR-Cas9 system in the past few years has made eukaryotic genome editing, and specifically gene knockout for reverse genetics, a simpler, efficient, and effective task. The system is directed to the genomic target site by a programmed single-guide RNA (sgRNA) that base-pairs with the DNA target, subsequently leading to site-specific double-strand breaks. However, many gene families in eukaryotic genomes exhibit partially overlapping functions and, thus, the knockout of one gene might be concealed by the function of the other. In such cases, the reduced specificity of the CRISPR-Cas9 system, which may lead to the cleavage of genomic sites that are not identical to the sgRNA, can be harnessed for the simultaneous knockout of multiple homologous genes. Here, we introduce CRISPys, an algorithm for the optimal design of sgRNAs that would potentially target multiple members of a given gene family. CRISPys first clusters all the potential targets in the input sequences into a hierarchical tree structure that specifies the similarity among them. Then, sgRNAs are proposed in the internal nodes of the tree by embedding mismatches where needed, such that the cleavage efficiencies of the induced targets are maximized. We suggest several approaches for designing the optimal individual sgRNA, and an approach that provides a set of sgRNAs that also accounts for the homologous relationships among gene-family members. We further show by in-silico examination over all gene families in the Solanum lycopersicum genome that our suggested approach outperforms simpler alignment-based techniques.Graphical abstractHighlightsMany genes in eukaryotic genomes exhibit partially overlapping functions. This imposes difficulties on reverse-genetics, as the knockout of one gene might be concealed by the function of the other.We present CRISPys, a graph-based algorithm for the optimal design of CRISPR systems given a set of redundant genes.CRISPys harnesses the lack of specificity of the CRISPR-Cas9 genome editing technique, providing researchers the ability to simultaneously mutate multiple genes.We show that CRISPys outperforms existing approaches that are based on simple alignment of the input gene family.

scholarly journals Unrestrained markerless trait stacking in Nannochloropsis gaditana through combined genome editing and marker recycling technologies

2018 ◽  
Vol 115 (30) ◽  
pp. E7015-E7022 ◽  
Author(s):  
John Verruto ◽  
Kristie Francis ◽  
Yingjun Wang ◽  
Melisa C. Low ◽  
Jessica Greiner ◽  
...  
Keyword(s):  
Genome Editing ◽  
Gene Knockout ◽  
Algal Species ◽  
Nuclear Genome ◽  
Gene Families ◽  
Concept Generation ◽  
Multiple Traits ◽  
Nannochloropsis Gaditana ◽  
Marker Recycling ◽  
New Genes

Robust molecular tool kits in model and industrial microalgae are key to efficient targeted manipulation of endogenous and foreign genes in the nuclear genome for basic research and, as importantly, for the development of algal strains to produce renewable products such as biofuels. While Cas9-mediated gene knockout has been demonstrated in a small number of algal species with varying efficiency, the ability to stack traits or generate knockout mutations in two or more loci are often severely limited by selectable agent availability. This poses a critical hurdle in developing production strains, which require stacking of multiple traits, or in probing functionally redundant gene families. Here, we combine Cas9 genome editing with an inducible Cre recombinase in the industrial alga Nannochloropsis gaditana to generate a strain, NgCas9+Cre+, in which the potentially unlimited stacking of knockouts and addition of new genes is readily achievable. Cre-mediated marker recycling is first demonstrated in the removal of the selectable marker and GFP reporter transgenes associated with the Cas9/Cre construct in NgCas9+Cre+. Next, we show the proof-of-concept generation of a markerless knockout in a gene encoding an acyl-CoA oxidase (Aco1), as well as the markerless recapitulation of a 2-kb insert in the ZnCys gene 5′-UTR, which results in a doubling of wild-type lipid productivity. Finally, through an industrially oriented process, we generate mutants that exhibit up to ∼50% reduction in photosynthetic antennae size by markerless knockout of seven genes in the large light-harvesting complex gene family.

scholarly journals Outcomes Comparison of SpCas9- and LbCas12a-mediated DNA Editing in Zebrafish Embryos

2020 ◽  
Author(s):  
Darya Meshalkina ◽  
Aleksei Glushchenko ◽  
Elana Kysil ◽  
Igor Mizgirev ◽  
Andrej Frolov
Keyword(s):  
Genome Editing ◽  
Mode Of Action ◽  
Target Gene ◽  
Gene Knockout ◽  
The Other ◽  
Zebrafish Embryos ◽  
Guide Rnas ◽  
Research Technology ◽  
Ribonucleoprotein Complexes ◽  
Dna Editing

CRISPR/Cas genome editing is a widely used research technology. Its simplest variant is gene knockout resulting from reparation errors after introduction of dsDNA breaks by Cas nuclease. We compared the outcomes of the break repair by two commonly used nucleases (SpCas9 and LbCas12a) in zebrafish embryos to reveal if application of one nuclease is advantageous in comparison to the other. To address this question, we injected ribonucleoprotein complexes of nucleases and corresponding guide RNAs in zebrafish zygotes and three days later sequenced the target gene regions. We found that LbCas12a breaks resulted in longer deletions and more rare inserts, in comparison to those generated by SpCas9, while the editing efficiencies of both nucleases were the same. On the other hand, overlapping protospacers were shown to lead to similarities in repair outcome, although they were cut by two different nucleases. Thus, our results indicate that the repair outcome depends both on the nuclease mode of action and on protospacer sequence.

scholarly journals Effects of knocking out three anthocyanin modification genes on the blue pigmentation of gentian flowers

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Keisuke Tasaki ◽  
Atsumi Higuchi ◽  
Aiko Watanabe ◽  
Nobuhiro Sasaki ◽  
Masahiro Nishihara
Keyword(s):  
Genome Editing ◽  
Hydroxy Group ◽  
Plant Species ◽  
Gene Knockout ◽  
The Other ◽  
Wild Type ◽  
Japanese Gentian ◽  
Flower Pigment ◽  
Mutant Lines

Abstract Genome editing by the CRISPR/Cas9 system has recently been used to produce gene knockout lines in many plant species. We applied this system to analyze Japanese gentian plants that produce blue flowers because of the accumulation of a polyacylated anthocyanin, gentiodelphin. Mutant lines in which anthocyanin modification genes were knocked out were examined to assess the contribution of each gene to the blue pigmentation of flowers. The targeted genes encoded anthocyanin 5-O-glycosyltransferase (Gt5GT), anthocyanin 3′-O-glycosyltransferase (Gt3′GT), and anthocyanin 5/3′-aromatic acyltransferase (Gt5/3′AT). The Gt5GT knockout lines accumulated delphinidin 3G, whereas the Gt3′GT knockout lines accumulated delphinidin 3G-5CafG as the major flower pigment. Knocking out Gt5/3′AT resulted in the accumulation of delphinidin 3G-5G-3′G and delphinidin 3G-5G as the primary and secondary pigments, respectively. These results indicated the existence of two pathways mediating the modification of delphinidin 3G-5G in flowers, with one involving a glycosylation by 3′GT and the other involving an acylation by 5/3′AT. The Gt5GT, Gt3′GT, and Gt5/3′AT transformants produced pale red violet, dull pink, and pale mauve flowers, respectively, unlike the vivid blue flowers of wild-type plants. Thus, the glycosylation and subsequent acylation of the 3′-hydroxy group of the B-ring in delphinidin aglycone is essential for the development of blue gentian flowers.

scholarly journals Draft genome of the Brazilian railroad worm Phrixothrix hirtus E.Olivier (Phengodidae: Coleoptera)

2021 ◽  
Author(s):  
Danilo Trabuco Amaral ◽  
Yasuo Mitani ◽  
Isabel Aparecida Silva Bonatelli ◽  
Ricardo Cerri ◽  
Yoshihiro Ohmiya ◽  
...  
Keyword(s):  
Gene Family ◽  
Genome Assembly ◽  
Red Light ◽  
Draft Genome ◽  
Gc Content ◽  
Gene Families ◽  
Neotropical Region ◽  
Comparative Genomic ◽  
List Type ◽  
Draft Genome Assembly

AbstractThe Neotropical region is the richest in bioluminescent Coleoptera species, however, its bioluminescence megadiversity is still underexplored in terms of genomic organization and evolution, mainly within the Phengodidae family. The railroad worm Phrixothrix hirtus is an important biological model and symbolic species due to its bicolor bioluminescence, being the only organism that produces true red light among bioluminescent terrestrial species. Here, we performed the partial genome assembly of P. hirtus, combining short and long reads generated with Illumina sequencing, providing an important source of genomic information and a framework for comparative genomic analyses for the evaluation of the bioluminescent system in Elateroidea. The estimated genome size has ∼3.4Gb, 32% of GC content, and 67% of repetitive elements, being the largest genome described in the Elateroidea superfamily. Several events of gene family expansions associated with anatomical development and morphogenesis, as well as distinct odorant-binding receptors and retrotransposable elements were found in this genome. Similar molecular functions and biological processes are shared with other studied species of Elateriformia. Common genes putatively associated with bioluminescence production and control, including two luciferase genes that displayed 7 exons and 6 introns, and genes that could be involved in luciferin biosynthesis were found, indicating that there are no clear differences about the presence or absence of gene families associated with bioluminescence in Elateroidea. In P. hirtus the conversion of L- to D-luciferin seems to involve additional steps using a Palmitoyl-CoA thioesterase instead of an Acyl-CoA synthetase, which was found in Lampyridae species.HighlightsFirst draft genome assembly of Phengodidae, the largest one described in Coleoptera;Gene family expansions associated with anatomical development and morphogenesis;Bioluminescent control and luciferin biosynthesis genes are common within Elateroidea;Despite similar bioluminescent system, metabolic routes may have evolved independently;

scholarly journals Identifying candidate detoxification genes in the ecdysteroid kinase-like (EcKL) and cytochrome P450 gene families in Drosophila melanogaster by integrating evolutionary and transcriptomic data

2020 ◽  
Author(s):  
Jack L. Scanlan ◽  
Rebecca S. Gledhill-Smith ◽  
Paul Battlay ◽  
Charles Robin
Keyword(s):  
Gene Family ◽  
Association Study ◽  
Significant Proportion ◽  
Gene Families ◽  
Cytochrome P450s ◽  
Ecological Niches ◽  
Gene Gain ◽  
List Type ◽  
Toxic Stress ◽  
P450 Gene

AbstractThe capacity to detoxify toxic compounds is essential for adaptation to the ecological niches of many organisms, especially insects. However, detoxification in insects is often viewed through the lens of mammalian detoxification research, even though the organ and enzyme systems involved have diverged for over half a billion years. Phosphorylation is a non-canonical phase II detoxification reaction that, among animals, occurs near exclusively in insects, but the enzymes responsible have never been cloned or otherwise identified. We propose the hypothesis that members of the arthropod-specific ecdysteroid kinase-like (EcKL) gene family encode detoxicative kinases. To test this hypothesis, we annotated the EcKL gene family in 12 species of Drosophila and explored their evolution within the genus. Many ancestral EcKL clades are evolutionarily unstable and have experienced repeated gene gain and loss events, while others are conserved as single copy orthologs. Leveraging multiple published gene expression datasets from D. melanogaster, and using the cytochrome P450s—a canonical detoxification family—as a test case, we demonstrate relationships between xenobiotic induction, detoxification tissue-enriched expression and evolutionary instability in the EcKLs and the P450s. We also found previously unreported genomic and transcriptomic variation in a number of EcKLs and P450s associated with toxic stress phenotypes using a targeted phenome-wide association study (PheWAS) approach. Lastly, we devised a systematic method for identifying candidate detoxification genes in large gene families that is concordant with experimentally determined functions of P450 genes in D. melanogaster. Applying this method to the EcKLs suggested a significant proportion of these genes play roles in detoxification, and that the EcKLs may constitute a detoxification gene family in insects. Additionally, we estimate that between 11–16 uncharacterised D. melanogaster P450s are strong detoxification candidates.HighlightsThe poorly characterised ecdysteroid kinase-like (EcKL) gene family is hypothesised to encode enzymes responsible for detoxification by phosphorylation in insects.An integrative ‘detoxification score’ method accurately categorises the known functions of a canonical detoxification family, the cytochrome P450s, and suggests many EcKLs are also involved in detoxification.A targeted phenome-wide association study finds novel associations between EcKL/P450 variation and a number of toxic stress phenotypes, such as two unlinked EcKL paralogs that are both associated with developmental methylmercury resistance.

scholarly journals Analysis of the chromosomal clustering of Fusarium-responsive wheat genes uncovers new players in the defence against head blight disease

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Alexandre Perochon ◽  
Harriet R. Benbow ◽  
Katarzyna Ślęczka-Brady ◽  
Keshav B. Malla ◽  
Fiona M. Doohan
Keyword(s):  
Map Kinases ◽  
Gene Families ◽  
Gene Clusters ◽  
Head Blight ◽  
Orphan Gene ◽  
Metabolic Genes ◽  
Eukaryotic Gene ◽  
Blight Disease ◽  
Genomic Regions ◽  
Eukaryotic Genomes

AbstractThere is increasing evidence that some functionally related, co-expressed genes cluster within eukaryotic genomes. We present a novel pipeline that delineates such eukaryotic gene clusters. Using this tool for bread wheat, we uncovered 44 clusters of genes that are responsive to the fungal pathogen Fusarium graminearum. As expected, these Fusarium-responsive gene clusters (FRGCs) included metabolic gene clusters, many of which are associated with disease resistance, but hitherto not described for wheat. However, the majority of the FRGCs are non-metabolic, many of which contain clusters of paralogues, including those implicated in plant disease responses, such as glutathione transferases, MAP kinases, and germin-like proteins. 20 of the FRGCs encode nonhomologous, non-metabolic genes (including defence-related genes). One of these clusters includes the characterised Fusarium resistance orphan gene, TaFROG. Eight of the FRGCs map within 6 FHB resistance loci. One small QTL on chromosome 7D (4.7 Mb) encodes eight Fusarium-responsive genes, five of which are within a FRGC. This study provides a new tool to identify genomic regions enriched in genes responsive to specific traits of interest and applied herein it highlighted gene families, genetic loci and biological pathways of importance in the response of wheat to disease.

scholarly journals Isolation and Characterization Of Rice R Genes: Evidence for Distinct Evolutionary Paths in Rice and Maize

Genetics ◽  
1996 ◽  
Vol 142 (3) ◽  
pp. 1021-1031 ◽  
Author(s):  
Jianping Hu ◽  
Beth Anderson ◽  
Susan R Wessler
Keyword(s):  
Gene Family ◽  
Gene Families ◽  
Chromosome 1 ◽  
R Gene ◽  
R Genes ◽  
Helix Loop Helix ◽  
Isolation And Characterization ◽  
Undetermined Function ◽  
Evolutionary Paths

Abstract R and B genes and their homologues encode basic helix-loop-helix (bHLH) transcriptional activators that regulate the anthocyanin biosynthetic pathway in flowering plants. In maize, R/B genes comprise a very small gene family whose organization reflects the unique evolutionary history and genome architecture of maize. To know whether the organization of the R gene family could provide information about the origins of the distantly related grass rice, we characterized members of the R gene family from rice Oryza sativa. Despite being a true diploid, O. sativa has at least two R genes. An active homologue (Ra) with extensive homology with other R genes is located at a position on chromosome 4 previously shown to be in synteny with regions of maize chromosomes 2 and 10 that contain the B and R loci, respectively. A second rice R gene (Rb) of undetermined function was identified on chromosome 1 and found to be present only in rice species with AA genomes. All non-AA species have but one R gene that is Ra-like. These data suggest that the common ancestor shared by maize and rice had a single R gene and that the small R gene families of grasses have arisen recently and independently.

scholarly journals Expression of a gene duplication encoding conserved sperm tail proteins is translationally regulated in Drosophila melanogaster.

1993 ◽  
Vol 13 (3) ◽  
pp. 1708-1718 ◽  
Author(s):  
M Schäfer ◽  
D Börsch ◽  
A Hülster ◽  
U Schäfer
Keyword(s):  
Drosophila Melanogaster ◽  
Gene Family ◽  
Translational Control ◽  
Germ Line ◽  
Gene Families ◽  
Homologous Gene ◽  
Male Sterile ◽  
Sperm Tail ◽  
Repetitive Motif ◽  
Carboxy Terminal

We have analyzed a locus of Drosophila melanogaster located at 98C on chromosome 3, which contains two tandemly arranged genes, named Mst98Ca and Mst98Cb. They are two additional members of the Mst(3)CGP gene family by three criteria. (i) Both genes are exclusively transcribed in the male germ line. (ii) Both transcripts encode a protein with a high proportion of the repetitive motif Cys-Gly-Pro. (iii) Their expression is translationally controlled; while transcripts can be detected in diploid stages of spermatogenesis, association with polysomes can be shown only in haploid stages of sperm development. The genes differ markedly from the other members of the gene family in structure; they do not contain introns, they are of much larger size, and they have the Cys-Gly-Pro motifs clustered at the carboxy-terminal end of the encoded proteins. An antibody generated against the Mst98Ca protein recognizes both Mst98C proteins in D. melanogaster. In a male-sterile mutation in which spermiogenesis is blocked before individualization of sperm, both of these proteins are no longer synthesized. This finding provides proof of late translation for the Mst98C proteins and thereby independent proof of translational control of expression. Northern (RNA) and Western immunoblot analyses indicate the presence of homologous gene families in many other Drosophila species. The Mst98C proteins share sequence homology with proteins of the outer dense fibers in mammalian spermatozoa and can be localized to the sperm tail by immunofluorescence with an anti-Mst98Ca antibody.

scholarly journals Genome-wide search and structural and functional analyses for late embryogenesis-abundant (LEA) gene family in poplar

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Zihan Cheng ◽  
Xuemei Zhang ◽  
Wenjing Yao ◽  
Kai Zhao ◽  
Lin Liu ◽  
...  
Keyword(s):  
Gene Family ◽  
Higher Plants ◽  
Abiotic Stress Tolerance ◽  
Gene Families ◽  
Regulatory Elements ◽  
Late Embryogenesis Abundant ◽  
Genome Wide ◽  
Lea Gene ◽  
Late Embryogenesis ◽  
Genome Wide Search

Abstract Background The Late Embryogenesis-Abundant (LEA) gene families, which play significant roles in regulation of tolerance to abiotic stresses, widely exist in higher plants. Poplar is a tree species that has important ecological and economic values. But systematic studies on the gene family have not been reported yet in poplar. Results On the basis of genome-wide search, we identified 88 LEA genes from Populus trichocarpa and renamed them as PtrLEA. The PtrLEA genes have fewer introns, and their promoters contain more cis-regulatory elements related to abiotic stress tolerance. Our results from comparative genomics indicated that the PtrLEA genes are conserved and homologous to related genes in other species, such as Eucalyptus robusta, Solanum lycopersicum and Arabidopsis. Using RNA-Seq data collected from poplar under two conditions (with and without salt treatment), we detected 24, 22 and 19 differentially expressed genes (DEGs) in roots, stems and leaves, respectively. Then we performed spatiotemporal expression analysis of the four up-regulated DEGs shared by the tissues, constructed gene co-expression-based networks, and investigated gene function annotations. Conclusion Lines of evidence indicated that the PtrLEA genes play significant roles in poplar growth and development, as well as in responses to salt stress.

scholarly journals Shoot Regeneration Is Not a Single Cell Event

Plants ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 58
Author(s):  
Patharajan Subban ◽  
Yaarit Kutsher ◽  
Dalia Evenor ◽  
Eduard Belausov ◽  
Hanita Zemach ◽  
...  
Keyword(s):  
Gene Family ◽  
Single Cell ◽  
Shoot Regeneration ◽  
Major Gene ◽  
Molecular Genetic ◽  
Plant Biotechnology ◽  
Gene Families ◽  
Regeneration Medium ◽  
Developmental Processes ◽  
Leaf Segments

Shoot regeneration is a key tool of modern plant biotechnology. While many researchers use this process empirically, very little is known about the early molecular genetic factors and signaling events that lead to shoot regeneration. Using tobacco as a model system, we found that the inductive events required for shoot regeneration occur in the first 4–5 days following incubation on regeneration medium. Leaf segments placed on regeneration medium did not produce shoots if removed from the medium before four days indicating this time frame is crucial for the induction of shoot regeneration. Leaf segments placed on regeneration medium for longer than five days maintain the capacity to produce shoots when removed from the regeneration medium. Analysis of gene expression during the early days of incubation on regeneration medium revealed many changes occurring with no single expression pattern evident among major gene families previously implicated in developmental processes. For example, expression of Knotted gene family members increased during the induction period, whereas transcription factors from the Wuschel gene family were unaltered during shoot induction. Expression levels of genes involved in cell cycle regulation increased steadily on regeneration medium while expression of NAC genes varied. No obvious possible candidate genes or developmental processes could be identified as a target for the early events (first few days) in the induction of shoot regeneration. On the other hand, observations during the early stages of regeneration pointed out that regeneration does not occur from a single cell but a group of cells. We observed that while cell division starts just as leaf segments are placed on regeneration medium, only a group of cells could become shoot primordia. Still, these primordia are not identifiable during the first days.

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