crisper case9的应用是第三代基因编辑技术，主要有以下几种：
crisper case9的出现为人类基因编辑提供了新的可能性，随着近年来分子生物学的快速发展。
它已广泛应用于许多领域，特别是在遗传疾病治疗、疾病相关基因的筛选和检测、肿瘤治疗、动植物转化以及病原微生物的预防等领域具有巨大的潜力，可以有效改善人类的生活质量。具体在以下几个方面应用：
1、遗传疾病的矫正
2、艾滋病毒的治疗
3、癌症治疗
4、RNA编辑
5、分子诊断
RISPR-Cas9定义
RISPR-Cas9是一种基因治疗法，这种方法能够通过DNA剪接技术治疗多种疾病。2017年3月，英国《自然·通讯》杂志发表一项遗传学重要研究成果，利用CRISPR-Cas9系统可拯救失明小鼠。
以上内容参考：CRISPR-Cas9 - 百度百科
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
文章结构简单整理如下：
############################################################
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。
############################################################
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
这张图后面还会需要用到
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.
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）。
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！）
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
感谢师兄DZ
https://www.jianshu.com/p/bfb34ba1050d
接上文，还是这篇文章。但是上篇文章只是简单的介绍了一下CRISPR-Cas9系统，以及它的一些原理和应用介绍。今天还是基于上次的结构来进行补充。会保留上篇文章中的一些关键信息，以帮助理解。
文章结构简单整理如下：
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。
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。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。
Cas9 offers several potential advantages over ZFNs and TALENs , including the ease of customization, higher targeting efficiency and the ability to facilitate multiplex genome editing. As custom ZFNs are often difficult to engineer, we will primarily compare Cas9 with TALEN.
Cas9只需要重新购买一对oligos 20-nt gRNA即可，可是TALEM却需要重新设计新的TALEN.
WT S. pyogenes Cas9 (SpCas9) is known to make a blunt cut between the 17th and 18th bases in the target sequence (3 bp 5′ of the PAM). Mutating catalytic residues in either the RuvC or the HNH nuclease domain of SpCas9 converts the enzyme into a DNA nicking enzyme. In contrast, TALENs cleave nonspecifically in the 12–24-bp linker between the pair of TALEN monomer-binding sites.
Cas9可以同时对对个目的基因进行编辑，只要联合使用相应的sgRNA即可。
我放英文原文的原因是：要么这里很重要，担心自己的理解不能完全阐明；要么就是，这里不是那么重要，放出来看看就行。
Cas要求唯一的PAM存在3′ of the 20-bp target sequence，每个同源的Cas9有唯一的PAM序列，而这个情况在人类中却不是那么严格，常常每8-12个bp就能找到一个。
另外很重要的就是脱靶效应。
PAM必须紧跟在20-nt对应的靶向基因的下游，结合前面所说，降低脱靶效应也至关重要。
(i) the 5′-NGG PAM for S. pyogenes Cas9 and (ii) the minimization of off-target activity
We provide an online CRISPR Design Tool ( http://tools.genome-engineering.org ) that takes a genomic sequence of interest and identifies suitable target sites. To experimentally assess off-target genomic modifications for each sgRNA, we also provide computationally predicted off-target sites (for a detailed discussion, see Box 1)
为了解决以上不足之处，张老师课题组提供了在线设计sgRNA的工具，并且也提供了关于脱靶位点的计算方法以及增加编辑效率的alternative strategy（using the D10A nickase mutant of Cas9 (Cas9n) along with a pair of sgRNAs）---该部分内容比较多，感兴趣可回原文查看
CRISPR设计工具提供了所需的所有寡核苷酸和引物的序列(i)制备sgRNA结构，(ii)分析目标修饰效率，(iii)评估潜在靶外位点的切割。值得注意的是，由于表达sgRNA的U6 RNA聚合酶III启动子更倾向于将鸟嘌呤(G)核苷酸作为其转录本的第一个碱基，因此在sgRNA的5 '端附加了一个额外的G，而20-nt引导序列并不以G开头(图4 b, c)。在极少数情况下，某些sgRNA可能由于未知的原因而无法工作因此，我们建议为每个位点设计至少两个sgRNA，并在预期的细胞类型中测试它们的效率。
Isolation of clonal cell lines with specific modifications is often desired. This can be achieved after transfection by isolating single cells through either FACS (Steps 54–65) or serial dilutions (Steps 66–70) , followed by an expansion period to establish a new clonal cell line. It is worth noting that cell types can vary substantially in their responses to single-cell isolation, and literature specific to the cell type of interest should be consulted.
使用流式分离或者连续稀释法得到单克隆。
（1）SURVEYOR nuclease assay.
（2）Plasmid- or ssODN-mediated HDR.
（3）Detection of indels or HDR by sequencing
感谢师兄DZ
