Visualizing Endogenous Synaptic Proteins in Living Neurons

The recently published method is based on the generation of disulfide-free “intrabodies”, a structure from the 10th fibronectin type III domain known as FingRs. These affinity molecules were fused to GFP for direct fluorescence miscroscopy. The FingRs do not need di-sulfite bonds and are therefore better folders in mammalian cells. Specifically, a library was screened with in vitro display to identify FingRs that bind two synaptic proteins, Gephyrin and PSD95. After the initial selection, the researchers from USC secondarily screened binders using a cellular localization assay to identify potential FingRs that bind at high affinity in an intracellular environment. As it turned out, only 10-20% of the original positive clones bind well inside the cells, suggesting this type of further screening was a critical step.

The expression of intrabody is transcriptionally regulated by the target protein through a ZFN-repressor fusion. This transcriptional control system matches the expression of the intrabody to that of the target protein regardless of the target’s expression level. This design virtually eliminates unbound FingR, resulting in very low background that allows unobstructed visualization of the target proteins. As result, the FingRs presented in this study enabled live cell visualization of excitatory and inhibitory synapses, and apparently without affecting neuronal function.

Technically, the reason to use in vitro mRNA display was required by the need to use a large library (>10exp12, beyond the limit of the more commonly used phase display) to find good binders. A similar visualization system can be established using more potent affinity domains such as the VHH single-domain antibodies that have only one, sometimes dispensable, di-sulfite bond. The VHH domain nanobodies can be more easily isolated from camelid animals. Another improvement to the visualization system can be made by using stronger, superresolution-ready FPs such as mNeonGreen or mMaple to enable single molecule imaging, which is particularly interesting for studying synapses and applied to the BRAIN initiative.

Gross et al. Neuron, June 2013, http://www.ncbi.nlm.nih.gov/pubmed/23791193

李承鹏: 该不相信历史，还是不相信爱情

1368年，当世第一大屌丝朱重八终于逆袭成功了。

    站在应天城高高的台上，这个开国皇帝、也是帝国最资深的叫化子，不能忘怀当年正是贪官污吏让他流离失所，父母差点死无葬身之地。他心如明镜，官场贪腐让横扫天下的大元瞬间崩溃。他下定决心：绝逼要弄死丫们这些贪官污吏。是的，绝逼！

    那一年，他四十岁。

    他 精力充沛，侦察和分析官员异常财务状况时像一部云计算机。他肃贪苛细，宣布凡贪污60两以上银子的官员将被剥皮揎草，杀得兴起，干脆下令“不足60两也杀 掉”。早年的坎坷使他对贪腐恨之入骨，按现在纪检的说法就是对贪腐“零容忍”。在已知的素材里，可以看到亲任政法委总书记的他抓过的一些案子：“收贿袜子 一双、鞋两双”、“书籍四本、衣服一件”、“围脖一个、网巾一个、圆口衣服一件”……这让他看上去像个收破烂的。

    一方面出于叫化 子护食的本性，另一方面，贫苦出身的他认为乱世就得用重典。杀、杀……他成立了亲军督尉府，就是后来人们在影视剧里很熟悉的锦衣卫。又启用检校，那些军 人、官员、太监甚至和尚的职能很像现在的纪检或者巡视组，四处打探官员的负面。一经查实或不经查实，就会迅速砍掉那些人头。

    他花 了十五年率兄弟们打江山，花了十五年杀掉五万贪官以稳住江山。可贪官越来越多，那时出现这样一种景象，官员正在庭上牛逼哄哄审问犯人时，忽然被一拨更加牛 逼哄哄的锦衣卫冲进来抓走，弄得下面跪着的犯人也莫名其妙。又由于官员已不够用，只好留用一些犯了事的官吏让其戴枷办公。主审的官员和被审的犯人一样戴着 枷锁，官员后面站着监视他的锦衣卫，一俟审毕，再把官员拖出去打板子。这景象，十分壮观。

    多年以后，我们看到同样可爱的景观：早 上在报纸上看到市委的李书记严厉批评官风不正，晚上就在微博得知书记已被纪委带走了；刚刚看到纪委的曾书记抓了贪官，不一会儿就因涉黑被“双规”……新近 的广州白云区肃贪，由于被立案查处的干部多达81名，更由于常务副区长、副区长及原区委书记在内的3名主要领导涉案落马，查处干部太多，导致开会人数凑不 够。

    中国的官场史，一部按了循环播放键的滥剧。

    还是让我们回到明朝。话说贪官屡抓不绝，往往早上抓了三 个，晚上又出现五个。资深叫化子决意祭出群众路线。那时还没有新闻联播，焦点访谈仅限于内阁，他就向全国普遍发行了《大诰三编》，在《民拿害民该吏三十 四》里，他不忿地宣布：那些官员都是傻逼，现在我要动员德高望重的老人和见义勇为的豪侠们来帮我举报官员。后来更规定：任何一个百姓可以直接冲进官府，捉 拿不满意的官员，当官的若敢阻拦，则“夷诛全族”。于是通往首都的路上，常见一群群老百姓押解官员前往南京的盛况，活像黄金周旅游，那些当官的甚至下跪向 百姓求饶……真是大快人心。

    群众路线够彻底，视觉上也有种大革命的波澜壮阔。可是官员们仍然贪污，变花样儿贪污。

    他 郁闷。而且新情况出现了，一方面全国书生们如过江之鲫报考公务员，另一方面，人人自危的京官们每天上朝前要站在家门与妻儿诀别，哪个亲戚欠了钱未还、房契 在哪儿、下一代往什么方向培养、小升初找谁走后门……谁也不敢肯定这天上班之后还能不能再回来。一些官员想辞官。不行，“奸贪小人诽谤朝廷”。

    这个桥段由来已久……多年以后，一个叫刘志军的大官隔着铁栅栏告诉女儿“千万别沾政治”，被官媒批评“中伤政治”。还有一个叫赵光华的四川小官因受不了维稳压力辞去了副镇长的职位，被当成反面教材，被迫上网发表声明。

    压力山大，明朝的一些官员很爱得抑郁症。有的真抑郁，有的装疯。那时并没有太多高楼以供官员跳下来，割静脉自杀的法子也还没有流行开来。但喜欢明史的朋友都知道，有个叫袁凯的监察御史为了保命就装疯，他装疯的办法很有创意：吃屎。

    从南派三叔小说的角度，六百多年后的官员就是六百多年前的转世僵尸。看，龙岩的镇长又上吊了。而之前，福建莆田市市长张国胜在办公楼跳楼自杀，天津市政协主席宋平顺自杀，洛阳公安局纪检书记张广生跳楼自杀，浙江高院副院长童兆洪在卫生间上吊自杀……

    好 吧，还是让我们回到明朝。朱元璋真心郁闷，俸禄虽被抱怨不够养师爷，但你们又为老百姓做了多少事呢？虽说杀掉一些官员是为铲除异己，但很多官员的确贪腐得 不成体统。听听那些刑罚：挖膝盖、抽肠、用开水淋再用铁刷子刷、铁钩把人吊起风干……什么《电锯惊魂》弱爆了。朱元璋奇怪得那张瓦刀脸快形成一个巨大的问 号：“法数行而辄犯，奈何？”。为了几个破钱，丫们不怕死么？

    满朝文武，没人告诉他“渴马守水，饿犬护食”这个道理。一个叫桂彦良的大臣却发表了忠诚却相当二百五的意见：“用德则逸，用法则劳。”陛下该鼓励道德，树立官场道德模范。

    朱 元璋深以为然，开动所有国家机器宣传道德，极品道德文章“八股”也在这时集大成了。他这么推理，把圣人思想像软件一样植入未来官员的脑子里，官员有道德 了，国家自然昌盛了。这个推理影响久远，连后来推翻它的“清”也效仿。甚至几百年后的红朝也号召“流着道德的血液”，公务员统考时圣人、道德、高尚、和谐 等字眼跃然纸上。未来公务员摇篮的大学，则有幸深刻传达了《关于加强和改进高校青年教师思想政治工作的若干意见》……

    朱元璋恨不得在所有官员脑门上纹上“道德”二字，可帝国的吏治仍糟透了，它总共的十六任皇帝中，不乏勤勉之人，可直到末代的崇祯亡国，回天无力。

    帝国只相信两样东西：一是道德，二是酷刑。可从逻辑上，如果道德对约束官员有用，要那些下三滥刑罚干什么，如果酷刑是灵丹妙药，乡试、殿试何不考《论剁去贪官手脚、耳鼻制成人棍置于酱缸对未来吏治的可持续性发展》，至少字面看上去更有震撼力。

    它 从未想到过“法制”、“宪政”这些东西，也不知道差不多在朱重八站在应天城高台上决定玩死贪官的时候，一个叫英国的地方有下议院了。它不知道，锦衣卫、东 厂、检校这些并不是监督，而是监视，而监视只会让贪官更狡猾、更坚定地朋党结私，形成连皇帝也撼不动的利益集团。

    总之，这个精心 设计的帝国亡了。在它亡之前，有一个被人忽略的情节：1583年，万历皇帝在会试时出了一道匪夷所思的题目：朕越励精图治，官场却越腐败、法纪越松懈，到 底是朕缺乏仁爱，还是太优柔寡断呢？在神圣的全国统考时居然出这样的题目，前无古人后无来者。可见万历皇帝对吏治真是愁死了，憋得把本该给贵妃娘娘的私房 话都剧透出来。

    多年以后，红朝拥有了八十一万纪检干部，平均一个纪检干部监视八个官员。这比大明的锦衣卫和检校还要多。外加中央 巡视组和四十五个中央督导组，就是八府巡按的套路。这些日子，有些官员听说巡视组督导组驾临，前列腺都吓得掉裤裆了，每天上班前，说不定也深情回眸一眼平 日爱搭不理的黄脸婆……最近我们常欣闻某某贪官“闪电落马”。可是，现在闪电了，过去的二十多年里干吗去了？亲爱的闪电侠们。

    虽然不再四书五经六艺，但要坚持三个xx八x八x，世界上最神奇的道德就是，刘志军刚跟女孩做了“红楼梦”，就向领导感言“中国梦”。和明朝的贪官别无二致，他们被抓之后总爱玩这感恩呵梦想呵这些调调，仿佛只是误入红尘，内心还是小清新的。

    不 同的是，明朝宁错杀一千，也不放走一个。当今是：诰命夫人被判死缓，银行行长被判死缓，法院院长被判死缓，公安局长被判死缓，刘志军果然也死缓。在争论是 否“废死”时，我们只好调侃“不是取消死刑，而是取消死缓”。但认真你就输了。就算回到大明，抽肠、凌迟、、开水淋再铁刷子刷、钩起来风干……把惩治贪官 基本具体成一个制作腌卤食品的过程，也不会吓跑贪官。

    大明朝不也就出了一个海瑞吗。

    何况海瑞也弄得大家不开心。一个清官无钱葬母，这就不是大时代，也不是小时代，是宵小时代。

    算 了，不说影视，说明朝……《万历十五年》开头，黄仁宇先生专门写到1588年事情，英国大破西班牙无敌舰队。他没有提到的是，整整一百年后，也就是 1688年，英国的光荣革命诞生了，原本也贪腐、朋党、专制、国内形势乱七八糟的英国开始君主立宪，聪明地用分权、宪政、监督等手法治理国家，成为一时世 界霸主。而此时，中国的政权已移交到了“清”，清仍延续覆灭的“明”的道德+酷刑，甚至还采用了一个辅助战术，“文字狱”。帝国从此走下坡路。所谓康乾盛 世是教科书上涂抹的口红，潜伏的危机，均被四库全书那些才子们用修辞手法轻轻抹去。

    然后大家都去看热播电视连续剧《铁齿铜牙纪晓岚》去了。靠，傻逼和绅。哇，牛逼纪晓岚。生生把历史看成了言情。

    几千年来，中国的官场从不缺肃贪，妓院最爱假装打扫内部卫生了，中国官员也是最爱讲道德，婊子最爱述说自己清纯的爱情。很多时候，我们被迫在既有那么多肃贪、又有那么多道德的逻辑矛盾里，相信，丽春院发生过梁山伯与祝英台的爱情故事。

    就像相信人民日报的大标题，死缓彰显了法治精神。其实无论斩立决和还是死缓，都与彰显法治精神一根腿毛关系都没有，只与圣上一拍脑门的力气大还是利益集团拖后腿的力气大，有至深关系。

    公元1644年，崇祯自杀前写下遗诏：“朕凉德藐躬，上干天咎，然皆诸臣误朕。”就是说，你们这些负心人，平时白养你们了，关键时刻一个都不见，是你们搞死我的呀。

    276年前，帝国的第一任皇帝站在城头上发誓要搞死那些奸臣，经过276年卓越的努力，帝国最后一任皇帝终于死在歪脖子树下，哀怨认为自己是被奸臣搞死了。

    看着这枚循环播放键，到底是该不相信历史，还是不相信爱情。

Genome Modification—a Practical Approach

http://blog.allelebiotech.com/2013/07/talengenome_editingcrisprcas/

The ability to modify genomes has always been fervidly sought after by molecular, developmental biologists and geneticists as it would provide them with the means for finding out what a particular piece of the genome may do in the biological process they are studying. The discovery of naturally existing P-element helped a generation of Drosophila geneticists and made the fruit fly a prime model system for gene function studies in the 80’s and 90’s. But P-elements inserted at uncontrolled sites, making it essentially a gene transfer vehicle without much control. The introduction of prokaryotic recombination systems, e.g. LoxP and Cre, provided researchers with tools to obtain more control of the inserted genes in a host chromosome during a biological process such as development. Transposons like Sleeping Beauty, Piggybac, or Tol2 made similar experiments possible in mammalian cells.

Still, the randomness of transposon-type elements’ insertion, much like retrovirus or lentivirus, could cause trouble if they land in an undesirable spot. Methods of inserting transgenes only in well-known, harmless, and transcriptionally active regions, so called “safe harbors”, were subjects of interest of researchers and NIH grant topics in the past couple of years under “directed genome editing”. Gene knock-out or knock-in can be achieved through vector-mediated homologous recombination such as the rAAV genome engineering system and the “TARGATT” system, which are commercially available as kits or services.

However, instead of inserting an exogenous gene, it is often highly desirable to modify an endogenous genome sequence, which requires the modification apparatus to first recognize the target sequence. ZFN and TALEN both recognize DNA targets through specific nucleotide binding protein domains, with TALEN having more flexibility if assembled in a “Lego”-like format because each domain can specifically recognize a “C”, “G”, “T”, or “A” base. The description of using CRISPR/cas system in a recent burst of publications opened up new ways of binding to specific DNA sequences and nicking or severing the dsDNA. This system does not require engineeredDNA binding domain assembly; instead, it uses a guide RNA to find the target DNA sequence to direct endonuclease, in a sense quite like RNAi. However, the enthusiasm about CRISPR/cas was somewhat dampened by a report last month in Nature Biotechnology that reported off-target effects of CRISPR/cas was much higher than ZFN and TALEN. Particularly, if mismatches are located in the 5’ portion of the guide RNA targeting sequence, they can be well tolerated up to 3 or 4, even 5 mismatches. Unfortunately this is also similar to the tolerance of the RNAi matching region outside the core 12-base region. The difference is: for RNAi, the off-target damage is temporary and ignorable if the extent is insignificant compared to the effects on the intended target while for CRISPR/cas, an off-target cut on the chromosome is permanent.

On the positive side, in an even more recent publication in Nature Methods, mutant strains of C. elegans were obtained using the CRISPR/cas system and no evidence was found for off-target changes, at least not in an overwhelming fashion. Much value of the estimates of off-target effects relates to the methods used for analysis. Currently, most of the studies looked at potential off-target sides by searching for partial matches. In the future, whole genome sequencing will be increasingly required for submitting such publications.

On a practical note, if you intend to take a dive and try to use any one of these methods, your number one problem will be that none of the methods will result in 100% modification even if you can ignore the off-target problems for now. Therefore, many of our customers ask about a screening strategy. One could use traditional drug selection and fluorescent protein (FP)-based sorting, but these can only help you find cells that are successfully transfected with the ZFN, TALEN, or CRISPR/cas expressing DNA molecules, not necessarily having the genome modification result. We have formulated the idea of inserting the target site into an FP-bearing plasmid as a surrogate target cutting indicator, and use another FP to track transfection of the TALEN plasmid. Nonetheless, in the end, PCR-amplifying the target region of the chromosome and doing either an enzymatic mismatch detection assay (e.g. T7 endonuclease) or sequencing is the only way to know for sure whether genome editing has occurred.