
English: 
Hi my name is Jennifer Doudna from UC Berkeley
and I'm here today to tell you about how we uncovered
a new genome engineering technology.
This story starts with a bacterial immune system
that means understanding how bacteria
fight off a viral infection.
It turns out that a lot of bacteria
have in their chromosome,
which is what you are looking at here
a sequence of repeats shown in these black diamonds
that are interspaced with sequences
that are derived from viruses
and these have been noticed by microbiologists
who were sequencing bacterial genomes but nobody knew
what the function of these sequences might be
until it was noticed that they tend to also occur

Chinese: 
大家好，我是来自加州大学伯克利分校的Jennifer Doudna.
	今天请让我来向你们介绍，我们是如何研发出最新的基因组编辑技术的
这项技术的研发最先来源于对细菌免疫系统的研究
	具体来说，就是去了解细菌是如何抵抗病毒侵染的
研究发现，在很多细菌的染色体中有很多的重复序列
	就是你在这里看到的这些黑色的六边形， 而这些重复序列又被来源于病毒的一些序列间隔开

English: 
with a series of genes that often encode proteins
that have homology to enzymes that do interesting things
like DNA repair.
So it was a hypothesis that this system
which came to be called CRISPR
which is an acronym for this type of repetitive locus
that these CRISPR systems could actually be
an acquired immune system in bacteria
that might allow sequences to be integrated
from viruses and then somehow used later
to protect the cell from an infection
with that same virus.
So this was an interesting hypothesis
and we got involved in studying this
in the mid 2000's right after the publication
of three papers that pointed out
the incorporation of viral sequences
into these genomic loci.
And so what emerged over the next several years
was that in fact these CRISPR systems

Chinese: 
这些序列最初由测序细菌基因组的微生物学家发现
	但是没有人知道这些序列的功能是什么
	后来人们发现这些序列中包含的基因能够编码一些蛋白质
	这些蛋白质似乎和一些重要的酶
	譬如参与DNA修复的酶同源相似度很高
目前的假设是，CRISPR是细菌的获得性免疫系统 
	我们用CRISPR来称呼我们研究的这个系统
	CRIPSR是这种重复序列位点的名字的首字母缩写
	研究发现CRIPSR能够将病毒的遗传序列整合到细菌自身中。
	然后这些遗传序列能保护细菌不受到相同病毒的再次侵染。
这是一个非常有意思的假设。在2000年代中叶，3篇论文发表指出病毒的遗传序列可以被整合到细菌基因组里
	在这之后我们也开始参与到这项研究中来

English: 
really are acquired immune systems in bacteria
so until this point no one knew that bacteria
could actually have a way to adapt
to viruses that get into the cell
but this is a way that they do it
and it involves detecting foreign DNA
that gets injected like shown in this example
from a virus that gets into the cell
the CRISPR system allows integration
of short pieces of those viral DNA molecules
into the CRISPR locus
and then in the second step
that is shown here as CRISPR RNA biogenesis
these CRISPR sequences are actually transcribed
in the cell into pieces of RNA
that are subsequently used together
with proteins encoded by the CAS genes
these CRISPR-associated genes
to form interfering or interference complexes

Chinese: 
而在接下来的数年间
	研究证明CRISPRs确实是细菌的获得性免疫系统。
	在此之前，谁都想不到
	细菌居然有办法可以适应侵染它们细胞内的病毒。
	而它们正是通过CRISPR来做到这一点的
就像这里所画的，这个过程包括了三个步骤：首先是细菌检测病毒注入其中的外源DNA
	CRISPR系统能够把这些来自病毒的小片段DNA整合到基因组内的CRISPR基因位点
	第二步是CRISPR的RNA合成

English: 
that can use the information in the form
of these RNA molecules to base pair
with matching sequences in viral DNA.
So a very nifty way that bacteria
have come up with to take their invaders
and turn the sequence information against them.
So in my own laboratory
we have been very interested for a long time
in understanding how RNA molecules
are used to help cells to figure out
how to regulate the expression of proteins
from the genome.
And so this seemed like also a very interesting
example of this and
we started studying the basic molecular mechanisms
by which this pathway operates.
And in 2011 I went to a scientific conference
and I met a colleague of mine,
Emmanuelle Charpentier who is shown in this picture
on the far left and Emmanuelle's lab

Chinese: 
这些CRISPR序列会被转录成RNA片段进入细胞
	而这些RNA分子又会和由cas基因编码的蛋白质一起作用
	cas基因是CRISPR相关的基因。它们编码的cas蛋白和CRISPR RNA分子能够形成一些复合体
	这些复合体利用CRISPR RNA和病毒的DNA分子进行碱基配对
所以正是通过这种巧妙的方法
	细菌通过利用病毒入侵者的序列信息来反过来对付敌人
在我自己的实验室里，我们长期感兴趣的课题是细胞中RNA分子的功能
	以及如何调控基因组中的蛋白质表达
而CRISPR系统正是一个这方面很有意思的例子
	因此我们开始研究这条信号通路中的基础分子机制

Chinese: 
2011年间，我在参加一个学术会议时见到了我的一个同行，Emmanuelle Charpentier
	就是这幅图中最左边的女性
	Emmanuelle的实验室进行微生物研究，她们感兴趣的是作为人类病原体的细菌
Emmanuelle当时在研究的一种细菌叫做化脓性链球菌
	这种细菌在人类中能够造成非常严重的感染
非常奇怪的是，在这些链球菌中的CRISPR系统中有一种别的生物没有的基因
	而这个基因能够编码一种叫做cas9的蛋白
	遗传学实验证明了
	化脓性链球菌的CRISPR系统需要这个叫做cas9的蛋白才能正常工作
但在当时没有人知道这个蛋白的功能是什么
所以我们一起从各自的实验室里招募人员来研究cas9蛋白的功能

English: 
works on microbiology problems and they are
particularly interested in bacteria
that are human pathogens.
She was studying an organism called
Streptococcus pyogenes which is a bacterium
that can cause very severe infections in humans
and what was curious in this bug was that it
has a CRISPR system and in that organism
there was a single gene encoding a protein
known as Cas9
that had been shown genetically to be required
for function of the CRISPR system
in Streptococcus pyogenes,
but nobody knew at the time what the function
of that protein was.
And so we got together and recruited
people from our respective research labs
to start testing the function of Cas9.
So the key people in the project
are shown here in the photograph
in the center is Martin Jinek
who is a postdoctoral associate in my own lab
and next to him in the blue shirt
is Kryztof Chylinski who was a student
in Emmanuelle's lab

English: 
and so these two guys together with
Ines Fonfara who is on the far right,
a postdoc with Emmanuelle
began doing experiments across the Atlantic
and sharing their data.
And what they figured out was that
Cas9 is actually a fascinating protein
that has the ability to interact with DNA
and generate a double stranded break
in DNA at sequences that match
the sequence in a guide RNA
and this slide what you are seeing
is that the guide RNA
and the sequence of the guide in orange
that base pairs with one strand
of the double helical DNA
and very importantly this RNA
interacts with a second RNA molecule
called tracr that forms a structure
that recruits the Cas9 protein
so those two RNAs and a single protein
in nature are what are required
for this protein to recognize
what would normally be viral DNAs

Chinese: 
这张照片里面的正是这个项目中的关键人员
	在这中间的是我实验室的博士后，Martin Jinek
	在他旁边穿蓝色T恤的是Emmanuelle实验室的学生， Krzyszt Chylinski
他们和站在右边的Emmanuelle实验室的博士后Ines Fonfara
	开始了跨越大西洋的共同研究，彼此分享各自的实验数据
他们发现cas9是一种非常神奇的蛋白质。它能够和DNA相互作用，并且在DNA上形成一个双链缺口
	缺口形成的DNA序列位点是通过一个能与之互补的向导RNA分子决定的
在这张幻灯片里你所看到的是就是一个向导RNA分子。向导RNA上橙色的序列部分可以和双链DNA中的一条单链互补配对
非常重要的是，向导RNA能够结合另一个tracrRNA分子并形成一个特殊结构
	这个结构可以让cas9蛋白结合过来

Chinese: 
因此在自然界中
	细胞只需要两个RNA分子和一个蛋白质分子就能够识别入侵的病毒DNA
	而这个蛋白质分子的功能就是切断（来自病毒的）双链DNA
所以在当我们了解到这个事实之后
	我们认为，如果我们能够设计一个更简单的系统：把两个RNA分子合并成一个RNA分子
	这样这个功能的实验就只需要一个蛋白质分子和一个向导RNA分子了。真难道不是很酷么？
	简单的来说就是把你刚刚看到的这两个RNA分子连接到一起
	变成一个单独的向导RNA
	于是Martin Jinek在实验室里构建了这个分子
	然后我们进行了一个简单的实验
	来证明我们是否做出了一个可以调控的DNA剪切酶

English: 
in the cell and the protein
is able to cut these up,
literally by breaking up the double helical DNA.
And so when we figured this out
we thought: wouldn't it be amazing
if we could actually generate a simpler system
than nature has done
by linking together these two RNA molecules
to generate a system that would be a single protein
and a single guiding RNA.
So the idea was to basically take
these two RNAs that you see on the far side
of the slide and then basically link them together
to create what we call
a single guide RNA.
So Martin Jinek in the lab
made that construct
and we did a very simple experiment
to test whether we truly had
a programmable DNA cleaving enzyme
and the idea was to generate short single guide RNAs

Chinese: 
我们的想法是制造出一个短小的向导RNA来识别DNA分子上的不同位点
	就像这张幻灯片上的这个环状DNA分子
	我们设计的向导RNA分子专门识别这些由红色线段所表示DNA序列
接下来的实验就是把这个质粒，也就是这个环状DNA分子
	和两种不同的限制性内切酶孵育
	第一个酶是SaII，它能够切开这幅图上的DNA分子非常上游的灰色区域中的序列
	而第二个位置则是由RNA介导的cas9去识别剪切这些不同的红色线段标记的位置
在这个非常简单的实验中
	我们把DNA分子和限制性内切酶放到一起孵育反应了一段时间
	然后我们得到了以下的实验结果
	这里你看到的这个DNA凝胶能够让我们分离不同的被切开的DNA分子

English: 
that recognize different sites in a circular DNA molecule
that you see here
and the guide RNAs were designed
to recognize the sequences shown by the red bars
in the slide and the experiment was then
to take that plasmid, that circular DNA molecule
and incubate it with two different restriction (or cutting) enzymes,
one called SalI which cuts
the DNA sort of upstream at the far end
of the DNA in this picture
in the grey box,
and the second site being directed
by the RNA-guided Cas9
at these different sites shown in red.
And a very simple experiment
we did this incubation reaction
with plasmid DNA and this is the result
and so this is what you are looking at
is an agarose gel
that allows us to separate
the cleaved molecules of DNA
and what you can see is that in each of these reaction lanes
we get a different sized DNA molecule released

English: 
from this doubly digested plasmid
in which the size of the DNA
corresponds to cleavage at the different sites
directed by these guide RNA sequences
indicated in red
so this was a really exciting moment
actually a very simple experiment that was
kingd of an “A ha!” moment
when we said we really have a programmable DNA cutting enzyme
and that we can program it with a short piece of RNA
to cleave essentially any double stranded DNA sequence
so the reason we were so excited
about an enzyme that can be programmed
to generate double stranded DNA breaks
at any sequence is because
there was a long standing set of experiments
in the scientific community that showed
that cells have ways of repairing double stranded DNA breaks
that lead to changes
in the genomic information in DNA
so this is a slide that shows that

Chinese: 
你可以看到在这块凝胶上不同的泳道中
	有被剪切了两次后生成的不同大小的DNA分子
	而不同大小的DNA分子的产生正是由于不同序列的向导RNA识别不同红色标示的序列位点后剪切而来的
这真是一个非常让人激动的时刻，让人觉得“啊哈”
	一个非常简单的实验证明了我们确实做出了一个可以调控的DNA剪切酶
	而我们只需要设计一个很短的RNA分子
	就可以操纵去剪切任何一个双链DNA分子序列
我们之所以对这个发现感到如此激动的原因是
	长久以来科学界一直在证明
	细胞可以通过双链DNA分子剪切
	来对基因组中DNA所包含的信息进行修改

Chinese: 
这张幻灯片所显示的是，在包括Cas9系统之内的，任何剪切酶在制造了DNA分子双链缺口后
	细胞都有两种方式去检测并修复这些断裂的DNA分子：左边的这个是非同源末端连接
	DNA分子的末端链接回到一起
	这种方式通常会在缺口的位置产生一些小片段的插入或删除
而在右边的这个则是另一种修复方式，叫做同源性修复
	这里供体DNA分子上和被剪切的地方包含相同的信息
	从而可以被整合到基因组断裂的区域并提供新的遗传信息

English: 
after a double stranded break is generated
by any kind of enzyme that might do this
including the Cas9 system
those double stranded breaks in a cell
are detected and repaired by two types of pathways
one on the left that involves
non-homologous end joining
which the ends of the DNA are chemically ligated
back together usually with introduction
of a small insertion or deletion
at the site of the break
and on the right hand side
is another way that repair occurs
through homology directed repair
in which a donor DNA molecule
that has sequences that match those
flanking the site of of the
double stranded break can be integrated
into the genome at the site
of the break to introduce new genetic information
into the genome
so this had given many scientists

English: 
the idea that if there were a tool
or a technology that allowed
scientists or researchers to introduce
double stranded breaks at targeted sites
in the DNA of a cell then together
with all of the genome sequencing data
that are now available we know the
whole genetic sequence of a cell
and if you knew where a mutation occurred
that causes a disease for example
you could actually use a technology like this
to introduce DNA that would fix a mutation
or generate a mutation
you might like to study in a research setting
so the power of this technology is
really the idea that we can now generate
these types of double stranded breaks
at sites that we choose as scientists
by programming Cas9 and then allow
the cell to make repairs that introduce
genomic changes at sites of these breaks
but the challenge was how to generate the breaks

Chinese: 
已经有多位科学家提出
	如果能够有技术可以在细胞DNA中指定的位点制造DNA双链缺口
	然后利用我们目前所掌握的通过基因组测序所得到的对细胞遗传序列的知识
	假如我们知道造成疾病的那些遗传突变位点，我们就可以用这样一个技术
	去导入DNA序列，从而修复那些基因组中那些导致疾病的遗传突变
这项技术威力是
	我们可以通过操作Cas9在细胞中制造DNA双链缺口
	然后让细胞在修复基因组的同时在剪切位点改变基因组信息

Chinese: 
但是首先我们面对的挑战就是如何能够制造出这样的DNA缺口
	已经有很多实验室使用了不同的策略来实现这个目的
我要给大家展示的是其中两个例子，一个是锌指核酸酶，另一个是TAL效应子
	这两种都是可操纵的制造DNA双链缺口的方法
	但它们都是建立在蛋白质识别DNA分子序列的基础上的
所以说这些蛋白质可以通过不能的序列组合
	来实现对不同DNA分子序列的识别
	但这项技术要求大量的蛋白质工程实验

English: 
in the first place and so a number
of different strategies had been produced
for doing this in different labs
most of them, and I'm going to show
two specific examples here
one called zinc finger nucleases
and the other TAL effector domains
these are both programmable ways
to generate double stranded breaks in DNA
that will rely on protein-based recognition
of DNA sequences so these are proteins
that are modular, and can be generated
in different combinations of modules
to recognize different DNA sequences
it works as a technology
but it requires a lot of protein engineering
to do so, and what is really exciting
about this CRISPR/Cas9 enzyme
is that it is a RNA programmed protein
so a single protein can be used for
any site of DNA where we
would like to generate a break
by simply changing the sequence

English: 
of the guide RNA associated with Cas9
so instead of relying on protein-based recognition
of DNA we're relying on
RNA-based recognition of DNA
as shown at the bottom so what this means
is that is just a system
that is simple enough to use
that anybody with basic molecular biology training
can take advantage of this system
to do genome engineering
and so this is a tool that really
I think, fills out an essential
and previously missing component
of what we could call biology's IT toolbox
that includes not only the ability
to sequence DNA and look
at its structure, we know about
the double helix since the 1950's
and then in the last few decades
it's been possible to use enzymes
like restriction enzymes
and the polymerase chain reaction
to isolate and amplify particular segments
of DNA and now with Cas9
we have a technology that enables

Chinese: 
而让人感到振奋的是，CRISPR/Cas9酶是通过RNA分子介导的蛋白
	这个蛋白用能对DNA上的任何位点进行剪切
	仅仅是通过改变向导RNA分子的序列就可以实现这个目标
所以比起以往的那些通过蛋白质来识别DNA序列的方法，我们主要依赖于通过RNA分子来识别DNA序列，就是最下面的这个方法
这就意味着这个系统非常简单
	任何有基础生物学训练背景的人都可以利用它来进行基因组编辑
我认为我们发现的这个DNA编辑方法正是从前生物技术工具箱里所欠缺的核心内容
	不仅仅是像以前那样我们只能够测序DNA或者去了解DNA的结构
	要知道我们在上世纪五十年代就已经知道了DNA的双螺旋结构
	在过去的20年间
	我们已经可以利用限制性内切酶和聚合酶
	去分离和扩增我们所需的DNA片段

Chinese: 
而现在利用Cas9技术，我们可以实现快速编辑基因组
	世界上的任何实验室都可以利用CRISPR来实现他们的实验
所以总结一下这个新技术就是
	这个系统需要两个重要的组成部分
	它依赖于RNA分子和DNA分子的碱基配对来识别特定的序列位点
	非常重要的是，这个系统的操作非常简单直接
	我们可以同时设计不同的RNA分子，然后利用Cas9蛋白
	在一个细胞中同时进行多个位点的剪切
	或者我们可以通过一步实验
	就将一个染色体上一个很大的区域从基因组中删除
所以这项技术极大的推动了生物和遗传领域的发展
	全世界不同实验室都在利用这个技术设计进行很多有趣且新颖的应用

English: 
facile genome engineering
that is available to labs around the world
for experiments they might want to do
and so this is a summary of the technology
of the 2-component system
it relies on RNA-DNA base paring
for recognition
and very importantly because of the way
that this system works it
is actually quite straight forward
to do something called multiplexing
which means we can program Cas9
with multiple different guide RNAs
in the same cell to generate
multiple breaks and do things
like cut out large segments of a chromosome
and simply delete them in one experiment.
And so this has led to a real explosion
in the field of biology and genetics
with many labs around the world
adopting this technology
for all sorts of very interesting
and creative kinds of applications
and this is a slide
that's actually almost out of date now
but just to give you a sense

Chinese: 
这张宣传海报基本上已经过时了
	但我在这里想告诉大家的是，我们在2012年发表了我们最先的工作
	在那时，只有很少的一部分科学研究是关于Cas9的
	包括结晶生物学领域在内，任何生物学领域中，对cas9的研究都很少
而从2013年开始至今，不同实验室利用这项基因组编辑技术发表的文章数量以难以置信的速度增长
作为一个基础生物学的研究人员
	我为一项基础研究能够实现一项技术
	并让大家进行不同的精彩的实验而感到非常高兴

English: 
of the way that the field
has really taken off
so we published our original work on Cas9
in 2012 and up until that point
there was very little research
going on on CRISPR biology anywhere
it was a very small field
and then you can see that
starting in 2013 and extending
until now there has been this
incredible explosion in publications
from labs that are using
this as a genome engineering technology
so it's been really very exciting for me
as a basic scientist to see what started
as a fundamental research project
turned into a technology that turns out
to be very enabling for all sorts
of exciting experiments
and I just wanted to close by sharing
with you a few things
that are going on using this technology
so of course on the left hand side
lots of basic biology that can be done now
with the engineering of model organisms
and different kinds of cell lines
that are cultured in the laboratory
to study the behavior of cells

Chinese: 
最后我要和大家分享一些正在利用这个技术进行的实验， 在左边你可以看到
	很多基础生物学研究可以通过这个技术在不同的模式生物
	和不同的细胞系中中进行基因编辑，从而来学习细胞的行为
	在植物和真菌的生物技术研发中
	Cas9技术实现了很多不同的工业应用
	这项技术在生物医学中也有很大的前景
	它能够为人类疾病提供新的治疗方案
	这不仅听起来让人激动而且其实现已近在眼前了

English: 
but also in biotechnology being able to
make targeted changes in plants
and various kinds of fungi that could be very
useful for different sorts of industrial applications
and then of course in biomedicine
with lots of interest in the potential
to use this technology as a tool
for really coming up with novel therapies
for human disease I think is something
that is very exciting and is really something
that is on the horizon already
and then this slide just really indicates
where I think we're going to see this going
in the future with a lot of interesting
and creative kinds of directions
that are coming along in different labs
both in academic research laboratories
but also increasingly in commercial labs
that are going to enable the use of this
technology for all sorts of applications
many of which we couldn't even have
imagined even two years ago.

English: 
So very exciting and I want to just acknowledge a great team
of people that have been involved in working
on the project with me and we've
had terrific financial support from various groups
as well and it's been a pleasure
to share this with you, thank you.

Chinese: 
这里展示了在学术界中不同的实验室
	
	都在通过这项技术来实现很多有趣和有创新意义的新方向的研究
	而在商业实验室中
	利用这项技术进行的研究也在不断增加
	
	这在数年前都是很难想象到的
	而且让人感到非常振奋
最后我要感谢那些和我一起对这项技术做出杰出贡献的人员
	我还要感谢不同组织所给与的财力支持
	非常荣幸能和大家分享这个科学发现。谢谢大家
	
	Translation provided by Jinjin Zhu, Indiana University Bloomington
