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[学术文献 ] biGMamAct: efficient CRISPR/Cas9-mediated docking of large functional DNA cargoes at the ACTB locus 进入全文
Synthetic Biology
Recent advances in molecular and cell biology and imaging have unprecedentedly enabled multiscale structure-functional studies of entire metabolic pathways from atomic to micrometer resolution and the visualization of macromolecular complexes in situ, especially if these molecules are expressed with appropriately engineered and easily detectable tags. However, genome editing in eukaryotic cells is challenging when generating stable cell lines loaded with large DNA cargoes. To address this limitation, here, we have conceived biGMamAct, a system that allows the straightforward assembly of a multitude of genetic modules and their subsequent integration in the genome at the ACTB locus with high efficacy, through standardized cloning steps. Our system comprises a set of modular plasmids for mammalian expression, which can be efficiently docked into the genome in tandem with a validated Cas9/sgRNA pair through homologous-independent targeted insertion. As a proof of concept, we have generated a stable cell line loaded with an 18.3-kilobase-long DNA cargo to express six fluorescently tagged proteins and simultaneously visualize five different subcellular compartments. Our protocol leads from the in silico design to the genetic and functional characterization of single clones within 6 weeks and can be implemented by any researcher with familiarity with molecular biology and access to mammalian cell culturing infrastructure.
[政策法规 ] Regulatory Horizons Council: the Governance of Engineering Biology 进入全文
Regulatory Horizons Council(UK government)
Engineering biology (EB) is an overarching, agile and rapidly evolving technology platform with the potential to spawn transformative, or disruptive, innovations across a broad range of sectors of the economy. Its increasingly sophisticated range of genetic technologies, including genetic modification, synthetic biology, and genome editing, are enabling the creation of a new bioeconomy with the potential to disrupt or displace incumbent industries. The McKinsey Global Institute claims that “as much as 60 percent of the physical inputs to the global economy could, in principle, be produced biologically. Based on our interactions with stakeholders, we concluded that our most useful course of action would be to address the question, “How can we best manage overall governance of the complex array of products arising from the EB platform,” making recommendations on the most important issues and systemic interactions to be considered in providing future answers on a sector-by-sector basis or on the basis of novel classes of product. These recommendations are therefore intended to provide the framework for future decisions on EB governance, to be undertaken on a sectoral or market basis or based on common sets of properties.This report therefore addresses the challenges surrounding the effective governance of products of this broad range of enterprises. For regulators, there is an understandable desire for simplicity, to capture as many areas of application as possible under a single regulatory system; but that approach can result in the unnecessary failure of innovative developments that would otherwise contribute to growth of the UK economy, meeting Net Zero policy commitments or curing or preventing a disease. The challenge is to find an optimal balance between simplicity in governance systems and regulatory design, and their ability to handle a range of innovation scales and complexities. This report underscores the need for a dynamic, adaptive governance framework to support translation of EB products into markets and sets out recommendations for government that will help the UK to harness the potential of EB, contributing to economic growth, environmental sustainability, and public health."
[学术文献 ] Construction and iterative redesign of synXVI a 903 kb synthetic Saccharomyces cerevisiae chromosome 进入全文
nature communications
The Sc2.0 global consortium to design and construct a synthetic genome based on the Saccharomyces cerevisiae genome commenced in 2006, comprising 16 synthetic chromosomes and a new-to-nature tRNA neochromosome. In this paper we describe assembly and debugging of the 902,994-bp synthetic Saccharomyces cerevisiae chromosome synXVI of the Sc2.0 project. Application of the CRISPR D-BUGS protocol identified defective loci, which were modified to improve sporulation and recover wild-type like growth when grown on glycerol as a sole carbon source when grown at 37˚C. LoxPsym sites inserted downstream of dubious open reading frames impacted the 5’ UTR of genes required for optimal growth and were identified as a systematic cause of defective growth. Based on lessons learned from analysis of Sc2.0 defects and synXVI, an in-silico redesign of the synXVI chromosome was performed, which can be used as a blueprint for future synthetic yeast genome designs. The in-silico redesign of synXVI includes reduced PCR tag frequency, modified chunk and megachunk termini, and adjustments to allocation of loxPsym sites and TAA stop codons to dubious ORFs. This redesign provides a roadmap into applications of Sc2.0 strategies in non-yeast organisms.
[前沿资讯 ] CSHL and global collaborators map Solanum pan-genome 进入全文
Cold Spring Harbor Laboratory
CSHL researchers and colleagues around the globe have sequenced dozens of complete genomes for the plant genus that includes tomatoes, potatoes, and eggplants. In a process they deem “pan-genetics,” the biologists use their new, high-quality pan-genome to map the genes behind specific traits of agricultural significance across the genus, and target those genes to create desirable mutations. Their research reveals the importance of understanding the evolution of paralog genes—those that arise through gene duplication—in predicting genome editing outcomes. Importantly, their biggest breakthroughs didn’t come from plants in Lippman’s backyard. Instead, it was African eggplant. A tomato relative indigenous to the sub-Saharan region, African eggplant varies highly in fruit shape, color, and size.
[前沿资讯 ] AAFC research proving diploid breeding is more than just small potatoes 进入全文
Agriculture and Agri-Food Canada (AAFC)
Diploid potatoes are quickly becoming the stud spuds for breeding new commercial potato varieties. Wild diploid potatoes have 2 copies of each chromosome in their DNA, unlike more traditional tetraploid potatoes, like Russet Burbank, which have 4 copies of each chromosome. Fewer chromosomes make diploid genetics easier to work with for breeders. First domesticated in the Andes of South America, diploid potatoes are often a little smaller in size than their tetraploid cousins. Their untapped genetic potential is being harnessed by geneticists and breeders to develop new diploid potato varieties for farmers. Agriculture and Agri-Food Canada (AAFC) research scientist Dr. Bourlaye Fofana is leading the pre-breeding charge for diploid potatoes. After successfully pinpointing drought resistant and early maturing genes in diploid potatoes, he’s moved onto to his next foe — common scab disease. Common scab is a widespread issue that can lead to major economic losses for producers across Canada. Lost profits from waste occur when more than 5% of a potato is affected by unsightly common scab, which means they cannot be sold to the fresh vegetable market. Also, the lesions make potatoes difficult to peel, resulting in waste and lost profits in the chip and fry markets. Common scab resistant potato varieties are scarce, so developing potato varieties with resistance is vital for the industry. Wild diploid potatoes have 2 copies of each chromosome in their DNA make-up (genome), unlike more traditional tetraploid potatoes, like Russet Burbank, which have 4 copies of each chromosome. Fewer chromosomes make diploid genetics easier to work with and their untapped genetic potential is being harnessed by geneticists and breeders to develop new diploid potato varieties for farmers. Dr. Bourlaye Fofana, along with other AAFC researchers in Charlottetown, Prince Edward Island, and Kentville, Nova Scotia, as well as former AAFC student Dr. Braulio Soto-Cerda, now at the Universidad Católica de Temuco in Chile, evaluated 384 diploid potato clones for resistance to common scab disease. Of the 384 diploid clones, the group found 61 that are tolerant or resistant to common scab disease based on the percentage of the surface area of the potato covered by common scab, the severity of common scab, and the incidence of common scab. The 61 varieties will now be evaluated further for yield and quality as well as tested in larger commercial field plots to see how they perform in settings similar to farms.
[学术文献 ] The Silencing of the StPAM16-1 Gene Enhanced the Resistance of Potato Plants to the Phytotoxin Thaxtomin A 进入全文
MDPI
Potato common scab (CS) caused by Streptomyces scabiei is a severe disease that threatens tuber quality and its market value. To date, little is known about the mechanism regulating the resistance of potato to CS. In this study, we identified a presequence translocase-associated motor 16 gene from potato (designated StPAM16-1) that is involved in the response to the phytotoxin thaxtomin A (TA) secreted by S. scabiei. The StPAM16-1 protein was localized in the mitochondria, and the expression of the gene was upregulated in potato leaves treated with TA. The suppression of StPAM16-1 in potato led to enhanced resistance to TA and S. scabiei. Protein interaction analyses revealed that StPAM16-1 interacted with the subunit 5b of the COP9 signalosome complex (StCSN5). Similar to that of StPAM16-1, the expression levels of StCSN5 significantly increased in potato leaves treated with TA. These results indicated that StPAM16-1 acted as a negative regulator and was functionally associated with StCSN5 in the immune response of potato plants against CS. Our study sheds light on the molecular mechanism by which PAM16 participates in the plant immune response. Furthermore, both StPAM16-1 and StCSN5 could be potential target genes in the molecular breeding of potato cultivars with increased resistance to CS.
