The ability to edit plant genomes through gene targeting (
- NSF-PAR ID:
- 10077984
- Publisher / Repository:
- Wiley-Blackwell
- Date Published:
- Journal Name:
- Environmental Microbiology
- Volume:
- 21
- Issue:
- 6
- ISSN:
- 1462-2912
- Page Range / eLocation ID:
- p. 1942-1956
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
Summary GT ) requires efficient methods to deliver both sequence‐specific nucleases (SSN s) and repair templates to plant cells. This is typically achieved usingAgrobacterium T‐DNA , biolistics or by stably integrating nuclease‐encoding cassettes and repair templates into the plant genome. In dicotyledonous plants, such asNicotinana tabacum (tobacco) andSolanum lycopersicum (tomato), greater than 10‐fold enhancements inGT frequencies have been achieved usingDNA virus‐based replicons. These replicons transiently amplify to high copy numbers in plant cells to deliver abundantSSN s and repair templates to achieve targeted gene modification. In the present work, we developed a replicon‐based system for genome engineering of cereal crops using a deconstructed version of the wheat dwarf virus (WDV ). In wheat cells, the replicons achieve a 110‐fold increase in expression of a reporter gene relative to non‐replicating controls. Furthermore, replicons carryingCRISPR /Cas9 nucleases and repair templates achievedGT at an endogenousubiquitin locus at frequencies 12‐fold greater than non‐viral delivery methods. The use of a strong promoter to express Cas9 was critical to attain these highGT frequencies. We also demonstrate gene‐targeted integration by homologous recombination (HR ) in all three of the homoeoalleles (A, B and D) of the hexaploid wheat genome, and we show that with theWDV replicons, multiplexedGT within the same wheat cell can be achieved at frequencies of ~1%. In conclusion, high frequencies ofGT usingWDV ‐basedDNA replicons will make it possible to edit complex cereal genomes without the need to integrateGT reagents into the genome. -
A recently described symbiosis between the metabolically streamlined nitrogen‐fixing cyanobacterium
UCYN ‐A and a single‐celled eukaryote prymnesiophyte alga is widely distributed throughout tropical and subtropical marine waters, and is thought to contribute significantly to nitrogen fixation in these regions. SeveralUCYN ‐A sublineages have been defined based onUCYN ‐A nitrogenase (nifH ) sequences. Due to the low abundances ofUCYN ‐A in the global oceans, currently existing molecular techniques are limited for detecting and quantifying these organisms. A targeted approach is needed to adequately characterize the diversity of this important marine cyanobacterium, and to advance understanding of its ecological importance. We present findings on the distribution ofUCYN ‐A sublineages based on high throughput sequencing ofUCYN ‐AnifH PCR amplicons from 78 samples distributed throughout many major oceanic provinces. TheseUCYN ‐AnifH fragments were used to define oligotypes, alternative taxonomic units defined by nucleotide positions with high variability. The data set was dominated by a single oligotype associated with theUCYN ‐A1 sublineage, consistent with previous observations of relatively high abundances in tropical and subtropical regions. However, this analysis also revealed for the first time the widespread distribution of theUCYN ‐A3 sublineage in oligotrophic waters. Furthermore, distinct assemblages ofUCYN ‐A oligotypes were found in oligotrophic and coastally influenced waters. This unique data set provides a framework for determining the environmental controls onUCYN ‐A distributions and the ecological importance of the different sublineages. -
Abstract Across insect genomes, the size of the cytochrome P450 monooxygenase (
CYP ) gene superfamily varies widely.CYP ome size variation has been attributed to reciprocal adaptive radiations in insect detoxification genes in response to plant biosynthetic gene radiations driven by co‐evolution between herbivores and their chemically defended hostplants. Alternatively, variation inCYP ome size may be due to random “birth‐and‐death” processes, whereby exponential increase via gene duplications is limited by random decay via gene death or transition via divergence. We examinedCYP ome diversification in the genomes of seven Lepidoptera species varying in host breadth from monophagous (Bombyx mori ) to highly polyphagous (Amyelois transitella ).CYP ome size largely reflects the size of Clan 3, the clan associated with xenobiotic detoxification, and to some extent phylogenetic age. Consistently across genomes, familiesCYP 6,CYP 9 andCYP 321 are most diverse andCYP 6AB ,CYP 6AE ,CYP 6B,CYP 9A andCYP 9G are most diverse among subfamilies. Higher gene number in subfamilies is due to duplications occurring primarily after speciation and specialization (“P450 blooms”), and the genes are arranged in clusters, indicative of active duplicating loci. In the parsnip webworm,Depressaria pastinacella , gene expression levels in large subfamilies are high relative to smaller subfamilies. Functional and phylogenetic data suggest a correlation between highly dynamic loci (reflective of extensive gene duplication, functionalization and in some cases loss) and the ability of enzymes encoded by these genes to metabolize hostplant defences, consistent with an adaptive, nonrandom process driven by ecological interactions. -
Summary Spirodela polyrhiza is a fast‐growing aquatic monocot with highly reduced morphology, genome size and number of protein‐coding genes. Considering these biological features of Spirodela and its basal position in the monocot lineage, understanding its genome architecture could shed light on plant adaptation and genome evolution. Like many draft genomes, however, the 158‐Mb Spirodela genome sequence has not been resolved to chromosomes, and important genome characteristics have not been defined. Here we deployed rapid genome‐wide physical maps combined with high‐coverage short‐read sequencing to resolve the 20 chromosomes of Spirodela and to empirically delineate its genome features. Our data revealed a dramatic reduction in the number of therDNA repeat units in Spirodela to fewer than 100, which is even fewer than that reported for yeast. Consistent with its unique phylogenetic position, smallRNA sequencing revealed 29 Spirodela‐specific microRNA , with only two being shared withElaeis guineensis (oil palm) andMusa balbisiana (banana). CombiningDNA methylation data and smallRNA sequencing enabled the accurate prediction of 20.5% long terminal repeats (LTR s) that doubled the previous estimate, and revealed a high Solo:IntactLTR ratio of 8.2. Interestingly, we found that Spirodela has the lowest globalDNA methylation levels (9%) of any plant species tested. Taken together our results reveal a genome that has undergone reduction, likely through eliminating non‐essential protein coding genes,rDNA andLTR s. In addition to delineating the genome features of this unique plant, the methodologies described and large‐scale genome resources from this work will enable future evolutionary and functional studies of this basal monocot family. -
Summary Eliminating or silencing a gene's level of activity is one of the classic approaches developmental biologists employ to determine a gene's function. A recently developed method of gene perturbation called CRISPR‐Cas, which was derived from a prokaryotic adaptive immune system, has been adapted for use in eukaryotic cells. This technology has been established in several model organisms as a powerful and efficient tool for knocking out or knocking down the function of a gene of interest. It has been recently shown that CRISPR‐Cas functions with fidelity and efficiency in
Ciona robusta . Here, we show that in CRISPR‐Cas mediated genomic knock‐ins can be efficiently generated. Electroporating a tissue‐specific transgene driving Cas9 and a U6‐driven gRNA transgene together with a fluorescent protein‐containing homology directed repair (FP‐HDR) template results in gene‐specific patterns of fluorescence consistent with a targeted genomic insertion. Using the Tyrosinase locus to optimize reagents, we first characterize a new Pol III promoter for expressing gRNAs from theC. robusta Ciona H1 gene, and then adapt technology that flanks gRNAs by ribozymes allowing cell‐specific expression from Pol II promoters. Next, we examine homology arm‐length efficiencies of FP‐HDR templates. Reagents were then developed for targeting Brachyury and Pou4 that resulted in expected patterns of fluorescence, and sequenced PCR amplicons derived from single embryos validated predicted genomic insertions. Finally, using two differentially colored FP‐HDR templates, we show that biallelic FP‐HDR template insertion can be detected in live embryos of the F0 generation.savignyi