crispr基因编辑

by Josh McMenemy

乔什·麦克梅尼(Josh McMenemy)

用CRISPR编程基因组 (Programming the genome with CRISPR)

科学家如何借助计算机编辑基因组 (How scientists edit genomes with the help of computers)

CRISPR (pronounced “crisper”) is part of a bacterial immune system evolved to ‘remember’ and remove invading viral DNA.

CRISPR(发音为“ crisper”)是细菌免疫系统的一部分，进化为“记忆”并去除入侵的病毒DNA。

Its name is short for ‘Clustered Regularly Interspaced Short Palindromic Repeats’. But despite its mouthful of an acronym and complex biological origins, its engineering application is straightforward. To get started, there is only one protein you need to understand — Cas9.

它的名称是“聚簇有规律间隔的短回文重复序列”的简称。 但是，尽管它有一个缩写词和复杂的生物学渊源，但其工程应用却很简单。 首先，您只需要了解一种蛋白质-Cas9。

Cas9 searches for a specified DNA sequence and cuts it by breaking both strands of the DNA molecule. This protein is useful to researchers because they can ‘program’ it to target any DNA sequence. A sgRNA (‘single guide’ RNA) molecule determines the sequence that Cas9 binds to. RNA is a biological molecule similar to DNA, that can bind to proteins and DNA.

Cas9搜索指定的DNA序列，并通过折断DNA分子的两条链将其切割。 该蛋白质对研究人员有用，因为他们可以对其进行“编程”以靶向任何DNA序列。 sgRNA(“单向导” RNA)分子决定了Cas9结合的序列。 RNA是类似于DNA的生物分子，可以与蛋白质和DNA结合。

sgRNAs are short sequences with a constant region and variable region. The constant region attaches the sgRNA to the Cas9 protein. The variable region causes Cas9 to bind to the DNA sequence that complements it (see the diagram below).

sgRNA是具有恒定区和可变区的短序列。 恒定区将sgRNA连接到Cas9蛋白。 可变区使Cas9与与其互补的DNA序列结合(请参见下图)。

Making sgRNA is cheap and fast. This allows researchers to quickly set up a Cas9 experiment that cuts any DNA sequence. Well, not actually any sequence. There is a small constraint: the target sequence must be flanked by the correct PAM (protospacer adjacent motif) — a short sequence of DNA.

制作sgRNA既便宜又快速。 这使研究人员可以快速建立一个可切割任何DNA序列的Cas9实验。 好吧，实际上没有任何顺序。 有一个小的限制：目标序列的两侧必须是正确的PAM(与原间隔子相邻的基序)，即短的DNA序列。

Streptococcus pyogenes is an infectious species of bacteria. In the version of Cas9 it produces, the PAM motif is ‘NGG’, where N is any nucleotide (the ‘letters’ that make up DNA).

化脓性链球菌是细菌的传染性物种。 在它产生的Cas9版本中，PAM基序为“ NGG”，其中N为任何核苷酸(组成DNA的“字母”)。

Luckily, the motif ‘NGG’ occurs roughly once every 42 basepairs in the human genome. This mean that researchers can find a target site near almost every sequence of interest.

幸运的是，“ NGG”基序在人类基因组中大约每42个碱基对出现一次。 这意味着研究人员可以在几乎每个感兴趣的序列附近找到目标位置。

Depending on the experimental set up, these cuts in the DNA can either cause a random change or a precise change to the DNA sequence (more on this later).

根据实验设置，DNA的这些切割可能会导致DNA序列的随机变化或精确变化 (稍后会详细介绍)。

Before jumping into writing this program, I recommend studying the Cas9 diagram below.

在着手编写此程序之前，我建议您研究以下Cas9图。

Note that DNA and RNA have a directionality based on their chemical structure. One end of the molecule is referred to as the 5` (‘five-prime’) end, and the other is referred to as the 3` (‘three-prime’) end. This is important, because the sequences 5`— AGG — 3` is not the same as 3` — AGG — 5`.

请注意，DNA和RNA基于其化学结构具有方向性。 分子的一个末端称为5`(“五引数”)末端，另一末端称为3`(“三引数”)末端。 这很重要，因为序列5`–AGG_3`与3`_AGG_5`不同。

By convention, DNA and RNA sequences are assumed to be written 5` to 3` unless otherwise marked. Sequences read in the 5` — 3` direction are called ‘forward’ sequences. Sequences read the other way (3` — 5`) are called ‘reverse’ sequences. This is an arbitrary convention.

按照惯例，除非另有说明，否则假定DNA和RNA序列写为5`至3`。 沿5`-3`方向读取的序列称为“正向”序列。 以其他方式读取的序列(3`-5`)称为“反向”序列。 这是一个任意约定。

The diagram above shows an example of Cas9 bound when the PAM is on the reverse (bottom) strand.

上图显示了当PAM位于反向(底部)链上时与Cas9结合的示例。

您的第一个CRISPR程序 (Your first CRISPR program)

场景 (The scenario)

A scientist has a DNA sequence of interest and wants a list of all CRISPR targets contained in the sequence. Finding every target by hand is tedious and error prone.

一位科学家有一个感兴趣的DNA序列，想要该序列中包含的所有CRISPR靶标的列表。 手动找到每个目标很乏味且容易出错。

The scientist wants a simple program where they can input a DNA sequence and have all possible Cas9 target sites returned. The scientist would also like the cut position and PAM sequence for each target site.

科学家想要一个简单的程序，在其中他们可以输入DNA序列并返回所有可能的Cas9目标位点。 科学家还希望每个目标位点的切割位置和PAM序列。

EXAMPLE INPUT (from Figure 1): 'CCACGGTTTCTGTAGCCCCATACTTTGGATG'

EXAMPLE OUTPUT: [{    'cut_pos': 6,    'pam_seq': 'TGG',    'target_seq': 'GTATGGGGCTACAGAAACCG',    'strand': 'reverse'  }, {    'cut_pos': 22,    'pam_seq': 'TGG',    'target_seq': 'GTTTCTGTAGCCCCATACTT',    'strand': 'forward'  }]

First, how do we find CRISPR targets in the sequence? Remember that the Cas9 protein can bind anywhere there is a ‘NGG’ motif.

首先，我们如何找到序列中的CRISPR靶标？ 请记住，Cas9蛋白可以结合有“ NGG”基序的任何位置。

The first step is to loop through the sequence looking for matches. When the program finds a ‘NGG’ match, we want to subtract three positions from the start of the PAM site, since that is where Cas9 cuts the DNA.

第一步是遍历序列以查找匹配项。 当程序找到“ NGG”匹配项时，我们要从PAM站点的开头减去三个位置，因为这是Cas9切割DNA的位置。

Then, we want to record the twenty basepairs before the PAM as the target sequence. Sounds good?

然后，我们要记录PAM之前的20个碱基对作为目标序列。 听起来不错？

Well, the algorithm described above would actually miss about half of all CRISPR sites — because DNA is double stranded. This means if a ‘CCN’ is the sequence on the forward strand, then ‘NGG’ is the sequence on the reverse strand.

嗯，上述算法实际上会丢失所有CRISPR位点的大约一半-因为DNA是双链的。 这意味着，如果“ CCN”是正向链上的序列，那么“ NGG”是反向链上的序列。

The program must also search for ‘CCN’ using similar logic for the reverse strand.

该程序还必须使用与反向链相似的逻辑来搜索“ CCN”。

范例程序 (Example program)

并非所有CRISPR靶标都相等 (Not all CRISPR targets are equal)

When CRISPR was first catching on, researchers would often pull up a sequence on their computer and pick targets by hand. Designing the optimal sgRNA has now become much more complex. Below are brief introductions to this complexity.

CRISPR首次流行时，研究人员通常会在计算机上提取一个序列并用手选择目标。 设计最佳的sgRNA现在变得更加复杂。 以下是这种复杂性的简要介绍。

脱靶 (Off-targets)

Researchers soon realized that Cas9 would sometimes bind and cut at loci that did not exactly match the target sequence. These off-target cuts would cause unintended changes in a researcher’s experiment (or potentially a patient’s genome in the case of a therapy!)

研究人员很快意识到，Cas9有时会在与靶序列不完全匹配的基因座处结合并切割。 这些偏离目标的切割会导致研究人员的实验发生意外变化(在治疗的情况下可能导致患者的基因组发生变化！)

To design a good guide, a program must look at the entire genome (which is approximately 3 billion nucleotides for humans) to calculate an off-target score. Researchers have also recently engineered the Cas9 protein to have less off-target activity.

为了设计一个好的指南，程序必须查看整个基因组(人类大约30亿个核苷酸)以计算脱靶得分。 研究人员最近还对Cas9蛋白进行了改造，使其具有较少的脱靶活性。

昏死 (Knockout)

When Cas9 binds, it creates a cut by making a double strand break to the DNA molecule. Most of the time, a cell can repair this break through a biochemcial pathway (called non-homologous end joining, or NHEJ).

当Cas9结合时，它会通过使DNA分子发生双链断裂而产生切割。 大多数时候，细胞可以通过生物化学途径(称为非同源末端连接，或NHEJ)修复这种断裂。

This pathway is not always perfect, and sometimes when Cas9 cuts, the repair process makes a small insertion or deletion in the DNA sequence. In a protein-coding region of DNA, these small insertions and deletions cause a frameshift mutation — which will often disrupt the protein’s function.

该途径并不总是完美的，有时Cas9切割时，修复过程会在DNA序列中产生少量插入或缺失。 在DNA的蛋白质编码区域中，这些小的插入和缺失会导致移码突变—通常会破坏蛋白质的功能。

Researchers will often knockout a gene to figure out how a protein affects a specific cell function or phenotype. Creating a knockout edit adds extra constraints to the sgRNA design, because now the guide must land in the coding region of the gene.

研究人员通常会剔除一个基因，以弄清蛋白质如何影响特定的细胞功能或表型。 创建基因剔除编辑会给sgRNA设计增加额外的限制 ，因为现在该指南必须落在基因的编码区域。

编辑中 (Editing)

Instead of knocking out a gene, there are many times a scientist wants to make a precision edit. This is especially useful when trying to correct a disease causing a mutation. The best way to do this is still being researched. Most methods involve adding an extra donor piece of DNA.

除了敲除一个基因外，很多时候科学家还希望进行精确的编辑。 当试图纠正引起突变的疾病时，这特别有用。 最佳方法仍在研究中。 大多数方法涉及添加额外的供体DNA 。

目标得分 (On-target score)

Some sgRNA sequences will cause Cas9 to cut better than others. Researchers have compared cutting efficiency across thousands of Cas9 targets to create predictive models of a sgRNA’s cutting efficiency.

一些sgRNA序列会导致Cas9的切割效果更好。 研究人员已经比较了数千个Cas9靶标的切割效率 ，从而创建了sgRNA切割效率的预测模型。

Microsoft even supports an open source repository for ‘Machine Learning-Based Predictive Modeling of CRISPR/Cas9 guide efficiency’.

微软甚至支持开源存储库，以实现“基于机器学习的CRISPR / Cas9引导效率预测模型”。

其他CRISPR-Cas系统 (Other CRISPR-Cas systems)

Researchers have discovered CRISPR-Cas systems in other bacteria. These other systems have different PAMs.

研究人员在其他细菌中发现了CRISPR-Cas系统 。 这些其他系统具有不同的PAM。

最后的笔记 (Final notes)

Hope you learned something new! If you want to learn more about the biology, medical applications, commercial applications, or ethical implications of CRISPR-Cas genome engineering, then I recommend reading A Crack in Creation by Jennifer Doudna and Samuel Sternberg. Jennifer Doudna is one of the original discovers of CRISPR’s underpinnings.

希望你学到新东西！ 如果您想了解有关CRISPR-Cas基因组工程的生物学，医学应用，商业应用或伦理意义的更多信息，那么我建议阅读Jennifer Doudna和Samuel Sternberg撰写的《 创造中的裂缝》 。 詹妮弗·杜德纳(Jennifer Doudna)是CRISPR基础的最初发现之一。

关于作者 (About the Author)

I was previously an undergraduate researcher in the Gersbach Lab at Duke University, and I am currently a Software Engineer at a Synthego.

我以前在本科研究员Gersbach实验室在杜克大学，和我目前在一个软件工程师Synthego 。

翻译自: https://www.freecodecamp.org/news/programming-the-genome-with-crispr-bd567a214e2a/

crispr基因编辑