20171006 生命科学领域三篇文献updates：眼泪发电：光遗传学：CRISPR在植物中的应用（综述）

1Applied Physics Letters：科学家利用眼泪实现发电

      近日爱尔兰利默克里大学的研究团队,首次证实对人类眼泪中的溶菌酶施压,也可达到发电效果.

    据悉,参与研究的索利马尼教授表示:"我们是第一支团队使用溶菌酶晶体证实压电效应的存在."报道称,人类的泪液与鸡蛋的蛋白一样,蕴含大量蛋白质,研究团队发现当眼泪中的"溶菌酶"晶体受压时,会产生一种名为"压电效应"的化学反应,并可把溶菌酶化成电力.团队相信,日后可把技术应用于收集能量和生物医药等范畴.

    研究负责人赛尔德教授说:"利用晶体是检测压电效应的黄金定律."这次研究结果刊登于科学学术杂志《应用物理信件》.

ABSTRACT

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.

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2Science Advances: 光遗传学新工具--新型质子起动机

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.

光遗传学(Optogenetics)是一门较新的技术,运用光控制活体组织神经元或肌肉细胞,在神经科学研究领域具有广泛应用.这种方法极为精确,能通过开启或关闭特定的信息传递通路控制单个神经元.类似的,亦可用于部分逆转视力或听力,以及控制肌肉收缩.

    作为光遗传学的主要工具,光敏蛋白,被编辑插入细胞后会附着在细胞表面,当暴露在光线之下时将离子跨细胞膜移动.因此,一个改造后的神经元细胞的神经信号可被某个特定光脉冲激活或抑制,这取决于所使用的光敏蛋白.

    来自德国尤里希研究中心团队描述了一个名叫NsXeR的新蛋白工具,它属于异视紫红质(xenorhodopsin)类.光暴露下,能激活单个神经元,使其向神经系统发出信号.同理,也能激活肌细胞.

    因为受离子浓度变化影响,为了激活细胞,最好阻断钙离子运输.但是当蛋白非选择性地输送各种正离子(如Ca2+)时,可能出现不良副作用.

    这种新发现的蛋白能避免失控的钙离子运输:它是选择性的,只泵质子(H+)进入细胞.由于这种选择性,相比它的主要竞争对手"光敏通道蛋白(channelrhodopsin)"不能区分正离子的特点,它具有相当大的优势.一个正电荷离子进入一个兴奋的细胞,会让内外膜之间的表面张力减小.膜的去极化导致一个神经或肌肉冲动.如果只泵入质子,在引起冲动的同时还可以减少其他副作用.

此外,异视紫红质不依赖离子浓度,能可靠地把质子泵入或泵出细胞.而光敏通道蛋白只允许离子从高浓度向低浓度方向运输.

"目前我们已经掌握了该蛋白质如何工作的所有必要数据,这会成为我们优化改造光遗传学技术蛋白质工具参数的基础,"第一作者高级研究员Vitaly Shevchenko说.

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在植物中的应用

PLANTS IN THE CRISPR BioTechniques, Vol. 63, No. 3, September 2017, pp. 96–101

    从人体胚胎的基因编辑到多种疾病的靶向治疗,CRISPR/Cas9技术正不断登上头条.不过,这种技术的影响不仅仅限于生物医学研究,植物学家也在用CRISPR来研究植物功能、对抗疾病和提高产量.在最新一期的《BioTechniques》上,Sarah Webb介绍了植物中的CRISPR.

    转基因植物其实已经出现了很多年,但一直存在争议.近年来,生物学家一直在开发改变基因组的其他方法,以便补充传统的植物育种策略.在CRISPR出现之前,他们通常采用TALEN方法.不过,CRISPR/Cas9很快就超越了其他基因编辑技术.

    许多研究人员都有着相似的经历:几年前,他们同时启动TALEN和CRISPR项目,但CRISPR很快就搞定了.Donald Danforth植物科学中心的Becky Bart说,虽然这两种技术都能实现精确的编辑,但TALEN是复杂的蛋白质,每个突变需要新合成;CRISPR则不同,研究人员只需要开发新的向导RNA,因此既便宜又快捷.

培育美味水果

    长期以来,科学家通过挖掘天然的植物突变体或以随机诱变作为工具,来了解作物的基因功能.冷泉港实验室的Zach Lippman侧重于了解开花过程,特别是番茄及相关的茄科植物.CRISPR的出现增强了他的工作.通过破坏基因的编码序列并产生无功能的蛋白质,这些功能研究可以快速探索特定基因对开花过程的影响.

    有了CRISPR技术,研究人员也许还能以新的方式来驯化植物.Lippman指出,茄科的一些植物从未被驯化过,但结出十分美味的水果.这些水果可以在野外采到,但不适合在农场或花园种植,因为或许植物很大,但水果很小,或时间太长.现在,研究人员考虑修饰这些野生物种的某些基因,或改变基因表达的水平.(对于吃货而言,这真是一条好消息!)

抵御各种疾病

    对于辛苦劳作的农民来说,植物病原体的出现往往会让几个月的劳动成果付之一炬.虽然植物的免疫系统能够清除这些效应分子,但特定植物基因中的序列保守,可成为病原体攻击的目标.这样的序列一旦确定,就被称为易感基因.

在此,CRISPR提供了一个方便的工具,可以确定这些基因,并产生抗病植物.英国Sainsbury实验室的Sophien Kamoun及其同事最近就用CRISPR消除了番茄的一部分易感基因.这种非转基因的植物快速发育,能够完全抵御常见的白粉病.

    爱荷华州立大学的Bing Yang也是在植物中应用CRISPR的先锋,他的研究重点是水稻枯萎病.这种疾病在南亚和非洲肆虐,它与蔗糖转运蛋白SWEET基因的启动子结合,诱导易感性的产生.Yang利用CRISPR技术多次改变这些启动子,其效果相当于植物疫苗.佛罗里达大学的Nian Wang及其同事则成功改变了葡萄柚中的已知易感基因,以帮助植物抵御柑橘溃疡.

    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 

技术上的挑战

    当然,在植物中应用CRISPR仍然存在一些技术上的难题.去除DNA片段相对容易,但在特定位置改变序列或引入基因则不大容易.植物本身也带来难题.植物的细胞壁可能是个障碍,让基因编辑机制难以到达植物细胞.据Kamoun介绍,对棉花来说,这就是个难题.

在某些情况下,研究人员会使用农杆菌、病毒或质粒来敲开植物的大门.不过,最近出现了一些新的选择.杜邦先锋的一项新技术用基因枪将核糖核蛋白复合物导入植物细胞.因此,他们几乎可以转化任何品种的玉米.他们也将Cas9直接导入细胞,促使编辑过的体细胞直接形成胚状结构.

监管上的问题

即使大有潜力,但CRISPR编辑过的植物要想进入农田,还存在法律和监管上的障碍.目前,关于CRISPR/Cas9的专利,加州大学伯克利分校和Broad研究所在不断打官司,导致相关的知识产权相当混乱.于是,一些公司还是专注于TALEN,而另一些公司则采用meganuclease核酸酶.

此外,全球的监管机构也尚未确定如何监管这些编辑过的植物.Kamoun认为,一个关键的问题是监管部门是关注最终产品还是关注生产过程.欧洲监管机构往往侧重于过程,而美国监管机构则倾向于关注最终产品.与转基因不同,基因编辑的过程不大容易检测到.

    Legal and regulatory hurdles Blake Meyers from the Danforth Center has been using CRISPR/Cas9 to introduce small changes in plant microRNAs

在过去很长一段时间内,植物学家都没有工具来应用他们所学到的知识.现在有了CRISPR技术,他们可以开展更多的研究来真正了解所有基因在植物中的作用,以及如何调整和改善它们.未来,也许有着无限的可能.(

综述原文：

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.

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