近日愛爾蘭利默克裡大學的研究團隊,首次證實對人類眼淚中的溶菌酶施壓,也可達到發電效果.
據悉,參與研究的索利馬尼教授表示:"我們是第一支團隊使用溶菌酶晶體證實壓電效應的存在."報導稱,人類的淚液與雞蛋的蛋白一樣,蘊含大量蛋白質,研究團隊發現當眼淚中的"溶菌酶"晶體受壓時,會產生一種名為"壓電效應"的化學反應,並可把溶菌酶化成電力.團隊相信,日後可把技術應用於收集能量和生物醫藥等範疇.
研究負責人賽爾德教授說:"利用晶體是檢測壓電效應的黃金定律."這次研究結果刊登於科學學術雜誌《應用物理信件》.
Here, we present experimental evidence of the direct piezoelectric effect in the globular protein, lysozyme. Piezoelectric materials are employed in many actuating and sensing applications because they can convert mechanical energy into electrical energy and vice versa. Although originally studied in inorganic materials, several biological materials including amino acids and bone, also exhibit piezoelectricity. The exact mechanisms supporting biological piezoelectricity are not known, nor is it known whether biological piezoelectricity conforms strictly to the criteria of classical piezoelectricity. The observation of piezoelectricity in protein crystals presented here links biological piezoelectricity with the classical theory of piezoelectricity. We quantify the direct piezoelectric effect in monoclinic and tetragonal aggregate films of lysozyme using conventional techniques based on the Berlincourt Method. The largest piezoelectric effect measured in a crystalline aggregate film of lysozyme was approximately 6.5 pC N−1. These findings raise fundamental questions as to the possible physiological significance of piezoelectricity in lysozyme and the potential for technical applications.
2Science Advances: 光遺傳學新工具--新型質子起動機Liu Jie,Huang Juan,Guo Huan et al. The conserved and unique genetic architecture of kernel size and weight in maize and rice.[J] .Plant Physiol., 2017.
光遺傳學(Optogenetics)是一門較新的技術,運用光控制活體組織神經元或肌肉細胞,在神經科學研究領域具有廣泛應用.這種方法極為精確,能通過開啟或關閉特定的信息傳遞通路控制單個神經元.類似的,亦可用於部分逆轉視力或聽力,以及控制肌肉收縮.
作為光遺傳學的主要工具,光敏蛋白,被編輯插入細胞後會附著在細胞表面,當暴露在光線之下時將離子跨細胞膜移動.因此,一個改造後的神經元細胞的神經信號可被某個特定光脈衝激活或抑制,這取決於所使用的光敏蛋白.
來自德國尤裡希研究中心團隊描述了一個名叫NsXeR的新蛋白工具,它屬於異視紫紅質(xenorhodopsin)類.光暴露下,能激活單個神經元,使其向神經系統發出信號.同理,也能激活肌細胞.
因為受離子濃度變化影響,為了激活細胞,最好阻斷鈣離子運輸.但是當蛋白非選擇性地輸送各種正離子(如Ca2+)時,可能出現不良副作用.
這種新發現的蛋白能避免失控的鈣離子運輸:它是選擇性的,只泵質子(H+)進入細胞.由於這種選擇性,相比它的主要競爭對手"光敏通道蛋白(channelrhodopsin)"不能區分正離子的特點,它具有相當大的優勢.一個正電荷離子進入一個興奮的細胞,會讓內外膜之間的表面張力減小.膜的去極化導致一個神經或肌肉衝動.如果只泵入質子,在引起衝動的同時還可以減少其他副作用.
此外,異視紫紅質不依賴離子濃度,能可靠地把質子泵入或泵出細胞.而光敏通道蛋白只允許離子從高濃度向低濃度方向運輸.
"目前我們已經掌握了該蛋白質如何工作的所有必要數據,這會成為我們優化改造光遺傳學技術蛋白質工具參數的基礎,"第一作者高級研究員Vitaly Shevchenko說.
Abstract
Generation of an electrochemical proton gradient is the first step of cell bioenergetics. In prokaryotes, the gradient is created by outward membrane protein proton pumps. Inward plasma membrane native proton pumps are yet unknown. We describe comprehensive functional studies of the representatives of the yet noncharacterized xenorhodopsins from Nanohaloarchaea family of microbial rhodopsins. They are inward proton pumps as we demonstrate in model membrane systems, Escherichia coli cells, human embryonic kidney cells, neuroblastoma cells, and rat hippocampal neuronal cells. We also solved the structure of a xenorhodopsin from the nanohalosarchaeon Nanosalina (NsXeR) and suggest a mechanism of inward proton pumping. We demonstrate that the NsXeR is a powerful pump, which is able to elicit action potentials in rat hippocampal neuronal cells up to their maximal intrinsic firing frequency. Hence, inwardly directed proton pumps are suitable for light-induced remote control of neurons, and they are an alternative to the well-known cation-selective channelrhodopsins.
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3BioTechniques：CRISPR在植物中的應用
PLANTS IN THE CRISPR BioTechniques, Vol. 63, No. 3, September 2017, pp. 96–101
從人體胚胎的基因編輯到多種疾病的靶向治療,CRISPR/Cas9技術正不斷登上頭條.不過,這種技術的影響不僅僅限於生物醫學研究,植物學家也在用CRISPR來研究植物功能、對抗疾病和提高產量.在最新一期的《BioTechniques》上,Sarah Webb介紹了植物中的CRISPR.
轉基因植物其實已經出現了很多年,但一直存在爭議.近年來,生物學家一直在開發改變基因組的其他方法,以便補充傳統的植物育種策略.在CRISPR出現之前,他們通常採用TALEN方法.不過,CRISPR/Cas9很快就超越了其他基因編輯技術.
許多研究人員都有著相似的經歷:幾年前,他們同時啟動TALEN和CRISPR項目,但CRISPR很快就搞定了.Donald Danforth植物科學中心的Becky Bart說,雖然這兩種技術都能實現精確的編輯,但TALEN是複雜的蛋白質,每個突變需要新合成;CRISPR則不同,研究人員只需要開發新的嚮導RNA,因此既便宜又快捷.
培育美味水果
長期以來,科學家通過挖掘天然的植物突變體或以隨機誘變作為工具,來了解作物的基因功能.冷泉港實驗室的Zach Lippman側重於了解開花過程,特別是番茄及相關的茄科植物.CRISPR的出現增強了他的工作.通過破壞基因的編碼序列並產生無功能的蛋白質,這些功能研究可以快速探索特定基因對開花過程的影響.
有了CRISPR技術,研究人員也許還能以新的方式來馴化植物.Lippman指出,茄科的一些植物從未被馴化過,但結出十分美味的水果.這些水果可以在野外採到,但不適合在農場或花園種植,因為或許植物很大,但水果很小,或時間太長.現在,研究人員考慮修飾這些野生物種的某些基因,或改變基因表達的水平.(對於吃貨而言,這真是一條好消息!)
抵禦各種疾病
對於辛苦勞作的農民來說,植物病原體的出現往往會讓幾個月的勞動成果付之一炬.雖然植物的免疫系統能夠清除這些效應分子,但特定植物基因中的序列保守,可成為病原體攻擊的目標.這樣的序列一旦確定,就被稱為易感基因.
在此,CRISPR提供了一個方便的工具,可以確定這些基因,並產生抗病植物.英國Sainsbury實驗室的Sophien Kamoun及其同事最近就用CRISPR消除了番茄的一部分易感基因.這種非轉基因的植物快速發育,能夠完全抵禦常見的白粉病.
愛荷華州立大學的Bing Yang也是在植物中應用CRISPR的先鋒,他的研究重點是水稻枯萎病.這種疾病在南亞和非洲肆虐,它與蔗糖轉運蛋白SWEET基因的啟動子結合,誘導易感性的產生.Yang利用CRISPR技術多次改變這些啟動子,其效果相當於植物疫苗.佛羅裡達大學的Nian Wang及其同事則成功改變了葡萄柚中的已知易感基因,以幫助植物抵禦柑橘潰瘍.
An example of tomato mildew, one of the bacterial diseases that researchers are looking to eliminate in plants through the use of CRISPR/Cas9 technology技術上的挑戰
當然,在植物中應用CRISPR仍然存在一些技術上的難題.去除DNA片段相對容易,但在特定位置改變序列或引入基因則不大容易.植物本身也帶來難題.植物的細胞壁可能是個障礙,讓基因編輯機制難以到達植物細胞.據Kamoun介紹,對棉花來說,這就是個難題.
在某些情況下,研究人員會使用農桿菌、病毒或質粒來敲開植物的大門.不過,最近出現了一些新的選擇.杜邦先鋒的一項新技術用基因槍將核糖核蛋白複合物導入植物細胞.因此,他們幾乎可以轉化任何品種的玉米.他們也將Cas9直接導入細胞,促使編輯過的體細胞直接形成胚狀結構.
監管上的問題
即使大有潛力,但CRISPR編輯過的植物要想進入農田,還存在法律和監管上的障礙.目前,關於CRISPR/Cas9的專利,加州大學伯克利分校和Broad研究所在不斷打官司,導致相關的智慧財產權相當混亂.於是,一些公司還是專注於TALEN,而另一些公司則採用meganuclease核酸酶.
此外,全球的監管機構也尚未確定如何監管這些編輯過的植物.Kamoun認為,一個關鍵的問題是監管部門是關注最終產品還是關注生產過程.歐洲監管機構往往側重於過程,而美國監管機構則傾向於關注最終產品.與轉基因不同,基因編輯的過程不大容易檢測到.
Legal and regulatory hurdles Blake Meyers from the Danforth Center has been using CRISPR/Cas9 to introduce small changes in plant microRNAs在過去很長一段時間內,植物學家都沒有工具來應用他們所學到的知識.現在有了CRISPR技術,他們可以開展更多的研究來真正了解所有基因在植物中的作用,以及如何調整和改善它們.未來,也許有著無限的可能.(
綜述原文：
Abstract
Sarah Webb explores how researchers are using CRISPR/Cas9 to solve agricultural problems.
From gene-edited human embryos to disease-free pigs for donor organs, applications of CRISPR/Cas9 technology are filling the headlines. But the impact of this gene-editing technique isn’t limited to biomedical research: Plant biologists are also using CRISPR to study molecular mechanisms underlying plant function, fight disease, and enhance plant productivity.
"The CRISPR craze has pretty much swept through plant biology," says Dan Voytas of the University of Minnesota. "I would say most groups doing plant gene editing are using CRISPR or similar reagents." As a result, CRISPR/Cas9 could prove pivotal in addressing the challenge of feeding the world’s growing population, which is expected to approach 10 billion by 2050.
New plant breeding
Transgenic plants (also know as genetically modified organisms or GMOs) have been around for decades. But the insertion of foreign genes and DNA to produce desirable traits has prompted controversy as well as rejection of these plants by some consumers. In recent years, biologists have been developing more tailored methods for altering genomes that complement traditional plant breeding strategies and dovetail with new genetic tools. Until the advent of CRISPR within the past 5 years, one of the more promising gene-editing technologies was TAL effector nucleases (TALENs), which were developed from building blocks that occur naturally in plants.
However, CRISPR/Cas9 has largely overtaken other gene-editing techniques. Researchers tell similar stories: A few years ago, they started working on projects using both TALENs and CRISPR/Cas9 side-by-side, but quickly settled on CRISPR. While both techniques offer precise editing, TALENs are large, complex proteins that must be newly synthesized for each mutation, says Becky Bart of the Donald Danforth Plant Science Center in St. Louis. But using CRISPR/Cas9, a researcher needs only to develop new guide RNAs, she says, and "very quickly you can test a bunch of constructs right in the lab." As a result, CRISPR is both cheaper and faster, says Bing Yang of Iowa State University. And combining CRISPR with a traditional plant breeding program offers the most potential for making precise changes quickly.
That doesn’t mean TALENs and other methods are completely out of the picture though. With the continuing uncertainty surrounding the patents and licensing of CRISPR technology, many companies are still centering their work around technologies such as TALENS and meganucleases, where the intellectual property rights are clear, says Voytas, who was one of the early developers of TALENs and is the Chief Science Officer of Calyxt—a Minnesota-based plant gene-editing company focusing on that technology (see "Legal and regulatory hurdles" sidebar).
Mining mutations
Scientists have long mined natural plant mutants that show up in fields or used random mutagenesis as a tool for understanding gene function in crops. "Hopefully, you hit a gene; hopefully, you get a change in the phenotype of interest, the trait of interest, and then you try to pin down which gene is broken," says Zach Lippman of Cold Spring Harbor Laboratory in New York. His laboratory focuses on understand the flowering process, particularly in tomatoes and the related Solanaceae (nightshade) family, so that they can ultimately manipulate the process to improve agriculture.
CRISPR has enhanced Lippman’s work. The power of the technology in plants, he says, is the ability to create guided chromosomal breaks in genes. By disrupting the coding sequences of genes and producing non-functional proteins, functional studies to look at the effects of specific genes on the flowering process are possible (1). "We can now use CRISPR to mutate those genes directly and in a very fast and efficient way, which was never before possible," he says.
Blake Meyers of the Danforth Center has been using CRISPR to introduce single-nucleotide changes in plant microRNAs in Arabidopsis. "It gives us a very powerful tool to make very small changes, kind of what we might think of as subtle changes that can have dramatic effects on processing," he says. He’s also been collaborating on a project with Yang where they』ve been looking at the impact of cutting out 70-kilobase chunks of the maize genome, which they can do with single-nucleotide precision. "With CRISPR-generated mutants, we can get anywhere from a single base, which can cause a frame-shift mutation, to multiples of three that give us in-frame deletions, to much larger deletions, all from the same original construct, and so it’s given us a lot of allelic diversity," he says.
But gene knockouts aren’t the only way CRISPR can be applied. Mammalian researchers have developed screening techniques using CRISPR with an inactivated Cas9 protein that can’t cleave DNA. Here, binding of the mutant Cas9 either activates or represses gene expression, serving as a type of dimmer switch, rather than simply turning expression on and off. This strategy could also be useful in plants. Many traits Lippman studies in tomatoes are quantitative reproductive traits, and a targeted technique that modulates gene expression could help tease out more details of the flowering and fruiting processes.
CRISPR technology is also allowing researchers to explore new ways to domesticate plants that have agricultural potential. "There are some species of plants in the Solanaceae family that make wonderful, edible fruits that have never been domesticated," Lippman notes. People eat the fruits collected in the wild, but they’re not suitable for farms or gardens because the plants might be large, but the fruits they produce are too small, or they might take too long to flower. However, with CRISPR, researchers can think about modifying genes in these wild species that are homologs or orthologs to tomatoes, or use a "dimming strategy" to change levels of gene expression.
"I think that’s very exciting because now you’re talking about creating new crops," Lippman says. But as some start thinking about cultivating new fruits, others are thinking about how to protect existing domesticated plants.
Stoking disease resistance
Plant pathogens, which deliver disease-causing molecules known as effectors to their hosts, can devastate a farmer’s crop, often causing financial ruin or food insecurity within a region. While the plant’s immune system works to clear these effector molecules (TAL effectors are are one example of these plant pathogen effectors), conserved sequences within specific plant genes can prove to be weak points, and the pathogen’s effectors can exploit them to cause disease. Once established within the plant’s genome, such sequences are known as susceptibility genes.
"If you remove the targets of effectors, then the pathogen would struggle in causing disease and modifying the plant to make it susceptible," explains Sophien Kamoun, who studies plant–pathogen interactions at the Sainsbury Laboratory in Norwich, United Kingdom. CRISPR offers a convenient tool for both identifying such genes and producing plants resistant to the disease. Kamoun and his colleagues recently removed a portion of a susceptibility factor in a tomato plant using CRISPR. The resulting non-transgenic plants, which were fully resistant to the fungal disease powdery mildew, were developed quickly, within 10 months (2).
Yang, who has been a key figure in the development of CRISPR technology in plants, is focused on bacterial blight in rice. This severe disease in South Asia and Africa takes advantage of binding to the promoter of sucrose transporter genes, SWEET genes, to induce susceptibility. Using CRISPR, Yang is able to make multiple changes to these promoters to produce the equivalent of a plant vaccine. Nian Wang and his colleagues at the University of Florida Citrus Research and Education Center have successfully modified yet another known susceptibility gene, for a bacterial disease citrus canker, in a species of grapefruit (3). They are currently looking for susceptibility genes in another destructive citrus disease, citrus greening, also known as Huanglongbing.
Bart, in collaboration with Voytas, is doing related research with cassava, a hearty tuberous root vegetable that serves as a food security crop in sub-Saharan Africa, South American, and Asia. Funded by the Bill and Melinda Gates Foundation, the two researchers and their team are looking at various mutations that would protect these plants from a bacterial disease and two viral diseases. To date, they』ve successfully screened for mutations that abolish susceptibility genes for two of the diseases, and they』ve regenerated plants with mutations that they』ll soon be testing for disease tolerance.
As with diseases in other organisms, pathogens are constantly adapting and changing. A disease that rapidly devastates a critical crop in one part of the world could lead to widespread famine, such as the Irish potato famine of the 1840s. CRISPR could provide a way to outpace those mutations or to generate plants with broad-spectrum resistance, according to Lippman. "Previously, if you had a disease that popped up and started to really knock out a crop or really hurt crop yields, you』d have to look for natural resistance that would exist in a wild species, or you』d have to use old-fashioned genetic engineering techniques, which may or may not work."Overcoming technical challenges
Some technical challenges remain in applying CRISPR in plants. Removing DNA snippets is relatively easy, but while changing sequences or introducing genes at specific locations are also possible, according to Kamoun, "It’s just not always easy." One challenge in plant biology, he says, will be to make other types of edits as routine as simple deletions.
Still other technical challenges come from the plant itself. The plant cell wall can be a formidable barrier to cross: In some plants it’s difficult or even impossible to get the gene-editing machinery into a plant cell. In cassava, there is a robust set of strategies for transforming the cells, Bart says. But she also works on cotton, a plant with no gene-editing options because transformation is so difficult.
In some cases, researchers might use Agrobacterium, viral delivery, or plasmid bombardment to deliver the gene-editing components. But recent innovations are overcoming some of these obstacles. A new technology from DuPont Pioneer uses a gene gun to blast ribonucleoprotein particles into plant cells. As a result, they can transform almost any variety of corn, Lippman says. The company can also deliver factors along with the Cas9 machinery that prompt edited somatic cells to directly form an embryo-like structure that can germinate into a tiny plant (4).
Skipping DNA entirely avoids another problem: The removal of the Cas9 enzyme after editing, which is a priority as such plants move toward the mainstream. With some crops, researchers can use conventional breeding strategies to segregate transgenes that include Cas9 for removal. Kamoun and his colleagues used this strategy in their tomato study. However, some plants, including cassava, don’t form seeds and are difficult to cross, which makes removing a transgene that encodes Cas9 more difficult.
Legal and regulatory hurdles Blake Meyers from the Danforth Center has been using CRISPR/Cas9 to introduce small changes in plant microRNAs (Click to enlarge)Legal and regulatory hurdles
Even with its potential, legal and regulatory hurdles remain before CRISPR-altered plants make it to farmer’s fields. Ongoing battles between the University of California at Berkeley and the Broad Institute of MIT and Harvard over the patents associated with CRISPR/Cas9 have led to muddiness surrounding intellectual property, says Voytas. His company, Calyxt, is focused on TALENs, while other companies have focused on meganucleases, where the commercial path forward is more certain.
In addition, agencies around the globe haven’t worked out how plants edited with these technologies will be regulated. Regulatory costs can crush small biotechnology companies trying to bring a product to market, says Blake Meyers, so they』d like to employ strategies that minimize or avoid those hurdles. A central issue may be whether regulatory agencies focus on the end product or on how it was made, says Sophien Kamoun. European regulatory agencies tend to focus on process, while U.S. regulators tend to focus on the end product.
Unlike transgenic crops, which introduce whole genes that remain within the organism, plant biologists can use breeding, segregation, and other strategies to eliminate the gene-editing machinery from plants. If researchers avoid transgenes, CRISPR-edited plants are often indistinguishable from plants that acquired genetic mutations naturally. The traceability question may prove important for regulation over the long term, Kamoun says. "Unlike transgenics you really don’t have a way to demonstrate that it went through the particular process of genomic editing."Regulatory guidance is evolving. The USDA recently held a comment period on new proposed guidelines, but so far the USDA has allowed more than 2 dozen transgene-free plant products with knockout mutations to move forward without regulatory oversight (5). "They』ve been very consistent in allowing people to go out into the field with those products," Voytas says. -- S.W.
Limitless possibilities
Despite the technical hurdles, CRISPR/Cas9 is changing plant biology as fast as it is revolutionizing other fields. Just a few years ago, a research article might have highlighted the ability to mutate plant genes using CRISPR, but now the title touts a better understanding of plant architecture, with CRISPR embedded in the Materials and Methods section. "I’m not saying that we don’t have a lot of work yet to do on technology development," Voytas says, but he adds that it’s satisfying to see this shift. "It’s become the tool and not the story."With the investments made by researchers and industry, Kamoun sees CRISPR-based gene-editing technology as maturing relatively rapidly in plants. "I think the challenge now becomes about finding the traits," he says. For a long time, plant biologists didn’t have the tools to apply the knowledge that they』d gained about interesting plants genes and then deliver those results to farmers. But now they have the technology, he says. "We need more research to actually understand what all of the genes are doing in plants and how we can tweak them and improve them."After years of mostly reading genomes, researchers are editing and moving toward rewriting those genomes in increasingly sophisticated ways, Voytas says. Synthetic biology, though rudimentary right now, could help modify plant genes to produce rare metabolites or even pharmaceuticals of interest. Such technologies could allow researchers to modify nutrient content to lower gluten levels in bread or optimize the fatty acid content in cooking oil. "The possibilities are limitless, but the editing allows us to start to harness and control those metabolic pathways," he concludes.
References
1.) Soyk, S.. 2017. Variation in the flowering gene SELF PRUNING 5G promotes day-neutrality and early yield in tomato. Nat Genet 49:162-168.
2.) Nekrasov, V.. 2017. Rapid generation of a transgene-free powdery mildew resistant tomato by genome deletion. Sci Rep 7:482.
3.) Jia, H.. 2017. Genome editing of the disease susceptibility gene CsLOB1 in citrus confers resistance to citrus canker. Plant Biotechnol J 15:817-823.
4.) Svitashev, S.. 2016. Genome editing in maize directed by CRISPR-Cas9 ribonucleoprotein complexes. Nat Commun 7:13274.
5.) Ledford, H. 2016. Gene-editing surges as US rethinks regulations. Nature 532:158-159.