Native Americans domesticated maize ( Zea mays ssp. mays ) from lowland teosinte parviglumis ( Zea mays ssp. parviglumis) in the warm Mexican southwest and brought it to the highlands of Mexico and South America where it was exposed to lower temperatures that imposed strong selection on flowering time. Phospholipids are important metabolites in plant responses to low-temperature and phosphorus availability and have been suggested to influence flowering time. Here, we combined linkage mapping with genome scans to identify High PhosphatidylCholine 1 ( HPC1 ), a gene that encodes a phospholipase A1 enzyme, as a major driver of phospholipid variation in highland maize. Common garden experiments demonstrated strong genotype-by-environment interactions associated with variation at HPC1, with the highland HPC1 allele leading to higher fitness in highlands, possibly by hastening flowering. The highland maize HPC1 variant resulted in impaired function of the encoded protein due to a polymorphism in a highly conserved sequence. A meta-analysis across HPC1 orthologs indicated a strong association between the identity of the amino acid at this position and optimal growth in prokaryotes. Mutagenesis of HPC1 via genome editing validated its role in regulating phospholipid metabolism. Finally, we showed that the highland HPC1 allele entered cultivated maize by introgression from the wild highland teosinte Zea mays ssp. mexicana and has been maintained in maize breeding lines from the Northern United States, Canada, and Europe. Thus, HPC1 introgressed from teosinte mexicana underlies a large metabolic QTL that modulates phosphatidylcholine levels and has an adaptive effect at least in part via induction of early flowering time.
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This content will become publicly available on February 15, 2025
Genome streamlining to improve performance of a fast-growing cyanobacterium Synechococcus elongatus UTEX 2973
Genome streamlining is an evolutionary strategy used by natural living systems to dispense unnecessary genes from their genome as a mechanism to adapt and evolve. While this strategy has been successfully borrowed to develop synthetic heterotrophic microbial systems with desired phenotype, it has not been extensively explored in photoautotrophs. Genome streamlining strategy incorporates both computational predictions to identify the dispensable regions and experimental validation using genome-editing tool, and in this study, we have employed a modified strategy with the goal to minimize the genome size to an extent that allows optimal cellular fitness under specified conditions. Our strategy has explored a novel genome-editing tool in photoautotrophs, which, unlike other existing tools, enables large, spontaneous optimal deletions from the genome. Our findings demonstrate the effectiveness of this modified strategy in obtaining strains with streamlined genome, exhibiting improved fitness and productivity.
more » « less- Award ID(s):
- 2037887
- NSF-PAR ID:
- 10492392
- Editor(s):
- Harwood, Caroline S.
- Publisher / Repository:
- ASM Journals
- Date Published:
- Journal Name:
- mBio
- ISSN:
- 2150-7511
- Subject(s) / Keyword(s):
- ["CRISPR-Cas3","genome minimization","cyanobacteria","large progressive deletion","essential gene identification"]
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Background Cas12a (formerly known as Cpf1), the class II type V CRISPR nuclease, has been widely used for genome editing in mammalian cells and plants due to its distinct characteristics from Cas9. Despite being one of the most robust Cas12a nucleases, LbCas12a in general is less efficient than SpCas9 for genome editing in human cells, animals, and plants.
Results To improve the editing efficiency of LbCas12a, we conduct saturation mutagenesis in
E. coli and identify 1977 positive point mutations of LbCas12a. We selectively assess the editing efficiency of 56 LbCas12a variants in human cells, identifying an optimal LbCas12a variant (RVQ: G146R/R182V/E795Q) with the most robust editing activity. We further test LbCas12a-RV, LbCas12a-RRV, and LbCas12a-RVQ in plants and find LbCas12a-RV has robust editing activity in rice and tomato protoplasts. Interestingly, LbCas12a-RRV, resulting from the stacking of RV and D156R, displays improved editing efficiency in stably transformed rice and poplar plants, leading to up to 100% editing efficiency inT 0plants of both plant species. Moreover, this high-efficiency editing occurs even at the non-canonical TTV PAM sites.Conclusions Our results demonstrate that LbCas12a-RVQ is a powerful tool for genome editing in human cells while LbCas12a-RRV confers robust genome editing in plants. Our study reveals the tremendous potential of these LbCas12a variants for advancing precision genome editing applications across a wide range of organisms.
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Abstract Intraspecific niche divergence is an important driver of species range, population abundance and impacts on ecosystem functions. Genetic changes are the primary focus when studying intraspecific divergence; however, the role of ecological interactions, particularly host‐microbiome symbioses, is receiving increased attention. The relative importance of these evolutionary and ecological mechanisms has seen only limited evaluation. To address this question, we used
Microcystis aeruginosa , the globally distributed cyanobacterium that dominates freshwater harmful algal blooms. These blooms have been increasing in occurrence and intensity worldwide, causing major economic and ecological damages. We evaluated 46 isolates ofM. aeruginosa and their microbiomes, collected from 14 lakes in Michigan, USA, that vary over 20‐fold in phosphorus levels, the primary limiting nutrient in freshwater systems. Genomes ofM. aeruginosa diverged along this phosphorus gradient in genomic architecture and protein functions. Fitness in low‐phosphorus lakes corresponded with additional shifts withinM. aeruginosa including genome‐wide reductions in nitrogen use, an expansion of phosphorus assimilation genes and an alternative life history strategy of nonclonal colony formation. In addition to host shifts, despite culturing in common‐garden conditions, host‐microbiomes diverged along the gradient in taxonomy, but converged in function with evidence of metabolic interdependence between the host and its microbiome. Divergence corresponded with a physiological trade‐off between fitness in low‐phosphorus environments and growth rate in phosphorus‐rich conditions. Co‐occurrence of genotypes adapted to different nutrient environments in phosphorus‐rich lakes may have critical implications for understanding howM. aeruginosa blooms persist after initial nutrient depletion. Ultimately, we demonstrate that the intertwined effects of genome evolution, host life history strategy and ecological interactions between a host and its microbiome correspond with an intraspecific niche shift with important implications for whole ecosystem function. -
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Abstract CRISPR‐Cas9 has been shown to be a valuable tool in recent years, allowing researchers to precisely edit the genome using an RNA‐guided nuclease to initiate double‐strand breaks. Until recently, classical RAD51‐mediated homologous recombination has been a powerful tool for gene targeting in the moss
Physcomitrella patens . However, CRISPR‐Cas9‐mediated genome editing inP. patens was shown to be more efficient than traditional homologous recombination (Plant Biotechnology Journal, 15, 2017, 122). CRISPR‐Cas9 provides the opportunity to efficiently edit the genome at multiple loci as well as integrate sequences at precise locations in the genome using a simple transient transformation. To fully take advantage of CRISPR‐Cas9 genome editing inP. patens , here we describe the generation and use of a flexible and modular CRISPR‐Cas9 vector system. Without the need for gene synthesis, this vector system enables editing of up to 12 loci simultaneously. Using this system, we generated multiple lines that had null alleles at four distant loci. We also found that targeting multiple sites within a single locus can produce larger deletions, but the success of this depends on individual protospacers. To take advantage of homology‐directed repair, we developed modular vectors to rapidly generate DNA donor plasmids to efficiently introduce DNA sequences encoding for fluorescent proteins at the 5′ and 3′ ends of gene coding regions. With regard to homology‐directed repair experiments, we found that if the protospacer sequence remains on the DNA donor plasmid, then Cas9 cleaves the plasmid target as well as the genomic target. This can reduce the efficiency of introducing sequences into the genome. Furthermore, to ensure the generation of a null allele near the Cas9 cleavage site, we generated a homology plasmid harboring a “stop codon cassette” with downstream near‐effortless genotyping. -
Pleiotropy, the concept that a single gene controls multiple distinct traits, is prevalent in most organisms and has broad implications for medicine and agriculture. The identification of the molecular mechanisms underlying pleiotropy has the power to reveal previously unknown biological connections between seemingly unrelated traits. Additionally, the discovery of pleiotropic genes increases our understanding of both genetic and phenotypic complexity by characterizing novel gene functions. Quantitative trait locus (QTL) mapping has been used to identify several pleiotropic regions in many organisms. However, gene knockout studies are needed to eliminate the possibility of tightly linked, non-pleiotropic loci. Here, we use a panel of 296 recombinant inbred advanced intercross lines of Caenorhabditis elegans and a high-throughput fitness assay to identify a single large-effect QTL on the center of chromosome V associated with variation in responses to eight chemotherapeutics. We validate this QTL with near-isogenic lines and pair genome-wide gene expression data with drug response traits to perform mediation analysis, leading to the identification of a pleiotropic candidate gene, scb-1 for some of the eight chemotherapeutics. Using deletion strains created by genome editing, we show that scb-1 , which was previously implicated in response to bleomycin, also underlies responses to other double-strand DNA break-inducing chemotherapeutics. This finding provides new evidence for the role of scb-1 in the nematode drug response and highlights the power of mediation analysis to identify causal genes.more » « less
