Genome engineering using the CRISPR-Cas9 system
最近要开始学习CRISPR-Cas9实验，对着动辄几十页的说明，实在是看不下去，不如就尝试用读书笔记的方式来学习吧。
今日要讲的当然是张峰老师组的protocol
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文章结构简单整理如下：
- abstract
- introduction
- material
- procedure
- anticipated results
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常见缩写及专业词汇：
- clustered regularly interspaced short palindromic repeats (CRISPR) ：就是一个剪短的成簇的短回文结构。
- DNA double-stranded breaks (DSBs) ：双链断裂，啊啊啊啊好疼啊！
- nonhomologous end joining (NHEJ)：无模板修复，引入indel，适合敲除
- homology-directed repair (HDR)：有模板修复，精准编辑。
- zinc-finger nucleases (ZFNs)：锌指核酸酶
- transcription activator-like effector nucleases (TALENs)：转录激活因子样效应物核酸酶
- single-stranded DNA oligonucleotides (ssODNs)：单链DNA寡核苷酸
- CRISPR RNA (crRNA) array
- Streptococcus pyogenes：化脓性链球菌
- single-guide RNA (sgRNA)
- guide RNA (gRNA)
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1. abstract
1.1 Targeted nucleases are powerful tools for mediating genome alteration with high precision. 照例说很需要强有力的基因编辑工具。
1.2 The RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system can be used to facilitate efficient genome engineering in eukaryotic cells by simply specifying a 20-nt targeting sequence within its guide RNA. 简单说由gRNA引导的Cas9核酸酶的有效性。
1.3 Here we describe a set of tools for Cas9-mediated genome editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, as well as generation of modified cell lines for downstream functional studies. 非同源连接(NHEJ)或同源定向修复(HDR)
1.4 To minimize off-target cleavage, we further describe a double-nicking strategy using the Cas9 nickase mutant with paired guide RNAs. 双切
This protocol provides experimentally derived guidelines for the selection of target sites, evaluation of cleavage efficiency and analysis of off-target activity. 本protocol提供了选择靶点的策略、评价切割的有效性和脱靶效应的分析。
1.5 Beginning with target design, gene modifications can be achieved within as little as 1–2 weeks, and modified clonal cell lines can be derived within 2–3 weeks. 从靶点设计开始，基因修饰可在1-2周内完成，而2-3周内可得到克隆细胞系。
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2. introduction（节选）
A number of genome editing technologies have emerged in recent years, including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the RNA-guided CRISPR-Cas nuclease system.
近年来出现的基因编辑技术：ZFNs（锌指核酸酶），TALENs（转录激活因子样效应物核酸酶），CRISPR-Cas核酸酶系统。
The first two technologies use a strategy of tethering endonuclease catalytic domains（连接内切酶催化域） to modular DNA-binding proteins for inducing targeted DNA double-stranded breaks (DSBs) at specific genomic loci. By contrast, Cas9 is a nuclease guided by small RNAs （在引导RNA的帮助下）through Watson-Crick base pairing with target DNA
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2.1 Precise genome editing using engineered nucleases--精确基因编辑
和ZFN，TALEN一样，CRISPR-Cas也是通过激活DSB的模式来达到基因标记的目的。
在CRISPR-Cas系统中，经Cas剪切形成DSB后，DNA可通过以下两种途径进行修复：（A）在缺乏修复模板的情况下，DSBs通过NHEJ过程重新连接，以插入/删除(indel)突变的形式留下疤痕，可用于基因敲除，indel的出现导致移码突变和终止密码子的过早出现。（B）在DNA修复模板的情况下，精确修复-可达到精确编辑的效果；修复模板可以是插入序列两侧带有同源臂的传统双链DNA靶向结构，也可以是单链DNA寡核苷酸(ssODNs)。
以下这句话很重要：Unlike NHEJ, HDR is generally active only in dividing cells, and its efficiency can vary widely depending on the cell type and state,
as well as the genomic locus and repair template.
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2.2 Cas9: an RNA-guided nuclease for genome editing--详细介绍Cas9
简介：CRISPR-Cas is a microbial adaptive immune system that uses RNA-guided nucleases to cleave foreign genetic elements. Three types (I–III) of CRISPR systems have been identified across a wide range of bacterial and archaeal hosts, wherein each system comprises a cluster of CRISPR-associated (Cas) genes, noncoding RNAs and a distinctive array of repetitive elements (direct repeats). These repeats are interspaced by short variable sequences derived from exogenous DNA targets known as protospacers, and together they constitute the CRISPR RNA (crRNA) array. Within the DNA target, each protospacer is always associated with a protospacer adjacent motif (PAM), which can vary depending on the specific CRISPR system。
CRISPR-Cas是细菌用来抵抗外来生物抵御系统。经过广谱检测，人们发现了三种主要的CRISPR系统，它们由CRISPR-associated (Cas)基因、非编码rna和一组独特的重复元素(直接重复)组成，而这些重复序列则由来自外源性DNA靶点(即原间隔体)的短可变序列直接间隔开来；重复序列+间隔序列=CRISPR RNA (crRNA) array。在有DNA靶点的情况下，每一个间隔序列都有一个前间区序列邻近基序（PAM）。
The Type II CRISPR system is one of the best characterized consisting of the nuclease Cas9, the crRNA array that encodes the guide RNAs and a required auxiliary trans-activating crRNA (tracrRNA) that facilitates the processing of the crRNA array into discrete units
II型CRISPR系统是最具特征的系统之一，它由核酸酶Cas9、编码引导rna的crRNA阵列和有助于将crRNA阵列加工成离散单元的所需辅助反式激活crRNA (tracrRNA)组成。
Cas9酶+（编码出gRNA的序列+反式激活crRNA，这两部分都是crRNA array的结果）
主要是crRNA有什么用，为什么要形成离散单元？
CRISPR RNA (crRNA) array，编码gRNA，再加上tracrRNA，则可达到定位+编辑的功能gRNA用于引导，tracrRNA用于结合靶点。
Furthermore, the crRNA and tracrRNA can be fused together to create a chimeric, single-guide RNA (sgRNA). Cas9 can thus be re-directed toward almost any target of interest in immediate vicinity of the PAM sequence by altering the 20-nt guide sequence within the sgRNA. 所以，人们就把crRNA和tracrRNA合在一起，成为了single-guide RNA，即sgRNA，而通过修改tracrRNA的序列，在理论上可以on-target任何目的靶点。
目前应用的经典例子： Direct injection of sgRNA and mRNA encoding Cas9 into embryos has enabled the rapid generation of transgenic mice with multiple modified alleles （获取基因工程鼠的好帮手！）
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在这里，我们详细解释了如何使用人类密码优化，核定位序列两侧野生型(WT) Cas9核酸酶或突变Cas9核酸酶促进真核细胞基因编辑。
We describe considerations for designing the 20-nt guide sequence, protocols for rapid construction and functional validation of sgRNAs and finally the use of the Cas9 nuclease to mediate both NHEJ- and HDR-based genome modifications in human embryonic kidney (HEK 293FT) and human stem cell (HUES9) lines
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