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基因组巡航导弹 精选

已有 9139 次阅读 2012-12-21 15:48 |个人分类:研究生培养|系统分类:科研笔记|关键词:学者| 基因组, 杂志, 巡航导弹

《科学》杂志公布今年的“2012年度十大科学突破”有一个内容是基因组的精密工程,不太理解,于是就找到《科学》杂志网页上看看更详细的介绍。以下根据个人理解整理的资料,以方便大家了解。个人的历史是,这一技术将来如果真的如预测的那样广泛使用,将会带来一场生物学技术上的革命。不仅对治疗一些基因性疾病,而且对研究许多基因的功能都具有深远的影响。

 

通常,人们无法确定对高级生物的DNA进行修改和删除的最终结果。然而,在2012年,名为“转录激活子样效应因子核酸酶”(TALENs)的工具赋予研究人员改变或关闭斑马鱼、蟾蜍、牲畜及其他动物甚至病人的细胞中特定基因的能力。这种技术以及其他新兴的技术与已有的基因靶向技术一样廉价和有效,同时它能让研究人员在健康人和病人中确认基因及变异的特定作用。

2012年,在基因工程研究者获得了多种新的功能强大的工具,生物学家可以在多种不同级别的物种使用这一手段。其中一种工具被称转录因子效应核酸酶(TALENs),TALENs具有破坏或改变斑马鱼、爪蟾和其他动物的特定基因的功能, TALENs是一种蛋白质可选择性切断DNA特定序列,能选择性修复特定基因(听上去好象是细菌的限制性内切酶)。其中一个小组利用这个技术用于小型猪心脏疾病模型的研究。其他一些小组使用这个技术对大鼠、蟋蟀甚至人类患者来源的细胞进行基因操作。晶体结构分析结果已经部分解释了这种蛋白是如何识别并结合DNA特定序列的机制,至少有三个小组已经找到制备这种蛋白的快速便宜的方法。这些进展将会促进这一领域的快速发展。

这一基因组操作技术在几年前是无法想到的,许多高等生物,改变或删除DNA序列具有极大的偶然性,成功率非常低。研究人员不太容易随意地插入新基因或删除旧基因,因此针对特定基因的操作以实现治疗基因性疾病是对人类的很大挑战。

十年前,锌指核酸酶的发现给人们提供了一种定位特定基因的技术,研究人员迅速开发了这种技术,但锌指核酸酶的制备十分困难,而且所有关键技术专利都被一个公司垄断。2009年,该领域再次出现突破。两个研究小组发现了可以和DNA重复系列进行一对一匹配结合的转录因子激活效应蛋白,或者说可以操作目标基因的特异蛋白。2012年,研究证明这种蛋白可以和锌指核酸酶功能类似,而且更容易实现,价格也更便宜。部分科学家甚至认为这一技术将成为分子生物学的标准技术。

 

两种手段的比较模式图

Comparison of zinc-finger nuclease (ZFN) and Transcription Activator-Like Effector Nuclease (TALEN) architecture. (a) ZFNs. Each ZFN polypeptide consists of two functional domains, a DNA-binding domain comprising a chain of finger modules (ZFs) that each typically recognize a unique 3-base pair sequence of DNA and a DNA-cleaving domain composed of the nuclease domain of the FokI nuclease. FokI functions as a dimer, hence when two FokI nucleases bind to DNA proximal to one another they can dimerize and introduce a double-strand break. Targeted double-strand DNA cutting can be obtained by designing zinc fingers for specific sequences that flank the desired cleavage site; in the example 12 base pairs per ZFN are targeted with polypeptides containing four zinc-finger modules each (ZF-1 through ZF-4 and ZF-5 through ZF-8). (b) Model of a TALEN. A TAL Effector (TALE) polypeptide contains a series of typically 34-amino acid repeats, of which residues 12 and 13 [repeat variable diresidues (RVDs) shown in orange] are responsible for recognition of a specific base as shown in the box (note that there is some discussion about the precision of the RVD NK recognition of G and other RVDs can specify base contacts61). FokI nuclease is fused to the C-terminal end of the protein using wild-type TALE sequence as a spacer. Several spacer lengths between the TALE binding core and FokI have demonstrated activity. The number of tandem 34-amino acid repeats in the binding core defines the length of the recognition sequence, and the end of the functional DNA-binding motif. Each target sequence must be preceded by a T nucleotide. Two TALENs are shown to assemble on a genomic sequence in the opposite polarity to ZFNs to form a heterodimeric cleavage complex.

 

传统的锌指核酸酶存在一定缺点,使用这种技术的难点是必须为每一个新的目标DNA序列重新设计蛋白类型,因为每个特定序列需要特殊的蛋白,这些锌指核酸酶由两个部分组成,DNA结合区和DNA切割区。这种TALEN新技术创造性地采用用RNA代替蛋白质的DNA结合区,这样可以使技术大大简化(制备RNA比蛋白质容易的多)。Cas9这是一种天然的细菌防御系统,具有切割DNA的功能。研究人员首先将两种RNAs融合,一种负责结合目标DNA,一种负责结合Cas9蛋白,利用这个系统,他们可以实现切除特定的目标DNA序列的目的。研究证明Cas9可以模拟TALEN的功能。现在科学家正尝试将这一系统应用到比细菌更复杂的生物体内。TALEN基因组操作系统是一种非常让人激动的手段,有希望代替锌指核酸酶使成为基因组操作的核心技术。《科学》杂志的年度评论中将这一技术称为基因组的巡航导弹技术,可见对这一技术寄予厚望。这个比喻十分恰当,在这个导弹系统中,RNA作为导航系统,而Cas9作为弹头。

Genomic Cruise Missiles

This year, genome engineers got their hands on some potentially powerful new tools that promise to put the modification of DNA within easy reach of biologists studying a variety of organisms, including yeast and humans. One of these tools, called TALENs (for “transcription activator–like effector nucleases”), can destroy or alter specific genes in zebrafish, Xenopus toads, and livestock. A TALEN is a protein that cuts DNA in specific places, and the ensuing repair modifies the target gene. One group of researchers used the technique to create a miniature pig useful for studying heart disease. Others are modifying the genomes of rats, crickets, and even human cells from patients with disease. Crystal structures of these effector proteins attached to DNA have revealed how the proteins find their targets. And at least three teams have come up with a way to make many of these proteins fast and cheaply. This progress has prompted more investigators to give this approach a try.

Such a boom in genome engineering was unthinkable just a few years ago. For most higher organisms, changing or deleting DNA has generally been a hit-or-miss proposition. Researchers could not readily control where an added gene would insert itself into a genome or which DNA they delete in so-called knockout experiments. As a result, pinpointing what specific genes do and correcting disease genes in people have posed major challenges.

 

CREDIT: COURTESY OF RECOMBINETICSA decade ago, a new technology called zinc finger nucleases provided a way to target specific genes. Researchers leaped to develop this tool. But zinc fingers proved difficult to make, and one company holds all the key patents. So excitement swelled again in 2009, when two teams discovered a one-to-one correspondence between the repetitive regions of transcription activator–like effector proteins and the DNA bases they attach to, thus providing a new way to target genes. In 2012, studies drove home that TALENs work as well as zinc fingers do but are far easier and cheaper to make. Some researchers now think TALENs will become standard procedure for all molecular biology labs.

Meanwhile, another gene-targeting technology is beginning to make a name for itself. One drawback of zinc finger nucleases, TALENs, and another genome-editing tool called meganucleases is that they must be reengineered for each new DNA target. These proteins have two parts: the DNA targeting section and the DNA-cutting section. The new technology substitutes RNA—which is simpler to make than a piece of a protein—for the DNA targeting section. It also makes use of a bacterial protein called Cas9, which is part of a natural bacterial defense system called CRISPR, to do the cutting.

Researchers have shown in a test-tube that they can combine these two RNAs into a single one that both matches the DNA target and holds Cas9 in place. Using this system, they were able to cut specific target DNA, demonstrating the potential of Cas9 to work like TALENs. Now, those researchers are trying this approach in organisms other than bacteria, and other genome engineers are quite excited about their prospects, suggesting that it may one day challenge zinc finger nucleases and TALENs as the core genome engineering technology.

 

 

Genome Engineering

A. N.-S. Mak et al., “The Crystal Structure of TAL Effector PthXo1 Bound to Its DNA Target,” Science 335, 716 (10 February 2012).

D. Deng et al., “Structural Basis for Sequence-Specific Recognition of DNA by TAL Effectors,” Science 335, 720 (10 February 2012).

D. F. Carlson et al., “Efficient TALEN-mediated Gene Knockout in Livestock,” PNAS 109, (23 October 2012).

D. Reyon et al., “FLASH Assembly of TALENs for High-throughput Genome Editing,” Nature Biotechnology30 460 (May 2012).

J. Kaiser, “Putting the Fingers On Gene Repair,” Science 310, 1894 (2005)

M. Jinek et al., “A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity,” Science 337 816-21 (17 August 2012).

V. M. Bedell et al., “In vivo Genome Editing Using a High-efficiency TALEN System,” Nature 491, 114 (1 November 2012).

Y. Lei et al., “Efficient Targeted Gene Disruption in Xenopus Embryos Using Engineered Transcription Activator-like Effector Nucleases (TALENs),” PNAS 109 (23 October 2012).

 



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