http://www.reibang.com/p/bfb34ba1050d
接上文琼掠，還是這篇文章。但是上篇文章只是簡(jiǎn)單的介紹了一下CRISPR-Cas9系統(tǒng)坛悉，以及它的一些原理和應(yīng)用介紹。今天還是基于上次的結(jié)構(gòu)來進(jìn)行補(bǔ)充。會(huì)保留上篇文章中的一些關(guān)鍵信息炕婶，以幫助理解。
Genome engineering using the CRISPR-Cas9 system
文章結(jié)構(gòu)簡(jiǎn)單整理如下：
- abstract--上文
- introduction--上文（部分）
- material
- procedure
- anticipated results
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常見縮寫及專業(yè)詞匯：
- clustered regularly interspaced short palindromic repeats (CRISPR) ：就是一個(gè)剪短的成簇的短回文結(jié)構(gòu)莱预。
- DNA double-stranded breaks (DSBs) ：雙鏈斷裂柠掂，啊啊啊啊好疼啊依沮！
- nonhomologous end joining (NHEJ)：無(wú)模板修復(fù)涯贞，引入indel，適合敲除
- homology-directed repair (HDR)：有模板修復(fù)危喉，精準(zhǔn)編輯宋渔。
- zinc-finger nucleases (ZFNs)：鋅指核酸酶
- transcription activator-like effector nucleases (TALENs)：轉(zhuǎn)錄激活因子樣效應(yīng)物核酸酶
- single-stranded DNA oligonucleotides (ssODNs)：?jiǎn)捂淒NA寡核苷酸
- CRISPR RNA (crRNA) array
- Streptococcus pyogenes：化膿性鏈球菌
- single-guide RNA (sgRNA)
- guide RNA (gRNA)
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1. abstract
2. introduction（節(jié)選）
2.1 Precise genome editing using engineered nucleases--精確基因編輯
和ZFN，TALEN一樣辜限，CRISPR-Cas也是通過激活DSB的模式來達(dá)到基因標(biāo)記的目的皇拣。
2.2 Cas9: an RNA-guided nuclease for genome editing--詳細(xì)介紹Cas9
CRISPR RNA (crRNA) array，編碼gRNA薄嫡，再加上tracrRNA氧急，則可達(dá)到定位+編輯的功能gRNA用于引導(dǎo)颗胡，tracrRNA用于結(jié)合靶點(diǎn)。所以态蒂，人們就把crRNA和tracrRNA合在一起杭措，成為了single-guide RNA，即sgRNA钾恢，而通過修改tracrRNA的序列手素，在理論上可以on-target任何目的靶點(diǎn)。
2.3 Comparison with other genome editing technologies--Cas和其他基因編輯手段對(duì)比（個(gè)人認(rèn)為很有必要補(bǔ)充這樣的背景知識(shí)瘩蚪，雖則貌似無(wú)用）
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.
2.3.1. Ease of customization--更易定制
Cas9只需要重新購(gòu)買一對(duì)oligos 20-nt gRNA即可泉懦，可是TALEM卻需要重新設(shè)計(jì)新的TALEN.
2.3.2. Cleavage pattern--剪切模式
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 nonspeci?cally in the 12–24-bp linker between the pair of TALEN monomer-binding sites.
2.3.3 Editing ef?ciency--編輯效率
Cas9可以同時(shí)對(duì)對(duì)個(gè)目的基因進(jìn)行編輯，只要聯(lián)合使用相應(yīng)的sgRNA即可疹瘦。
2.4 Limitations of the Cas9 system--不足之處
我放英文原文的原因是：要么這里很重要崩哩，擔(dān)心自己的理解不能完全闡明；要么就是言沐，這里不是那么重要邓嘹，放出來看看就行。
Cas要求唯一的PAM存在3′ of the 20-bp target sequence险胰，每個(gè)同源的Cas9有唯一的PAM序列汹押，而這個(gè)情況在人類中卻不是那么嚴(yán)格，常常每8-12個(gè)bp就能找到一個(gè)起便。
另外很重要的就是脫靶效應(yīng)棚贾。
2.5 Experimental design--實(shí)驗(yàn)設(shè)計(jì)
2.5.1 Target selection for sgRNA--sgRNA的選擇
PAM必須緊跟在20-nt對(duì)應(yīng)的靶向基因的下游，結(jié)合前面所說榆综，降低脫靶效應(yīng)也至關(guān)重要妙痹。
(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)
為了解決以上不足之處，張老師課題組提供了在線設(shè)計(jì)sgRNA的工具鼻疮，并且也提供了關(guān)于脫靶位點(diǎn)的計(jì)算方法以及增加編輯效率的alternative strategy（using the D10A nickase mutant of Cas9 (Cas9n) along with a pair of sgRNAs）---該部分內(nèi)容比較多怯伊，感興趣可回原文查看
CRISPR設(shè)計(jì)工具提供了所需的所有寡核苷酸和引物的序列(i)制備sgRNA結(jié)構(gòu)，(ii)分析目標(biāo)修飾效率判沟，(iii)評(píng)估潛在靶外位點(diǎn)的切割耿芹。值得注意的是，由于表達(dá)sgRNA的U6 RNA聚合酶III啟動(dòng)子更傾向于將鳥嘌呤(G)核苷酸作為其轉(zhuǎn)錄本的第一個(gè)堿基水评，因此在sgRNA的5 '端附加了一個(gè)額外的G猩系，而20-nt引導(dǎo)序列并不以G開頭(圖4 b, c)。在極少數(shù)情況下中燥，某些sgRNA可能由于未知的原因而無(wú)法工作;因此寇甸，我們建議為每個(gè)位點(diǎn)設(shè)計(jì)至少兩個(gè)sgRNA，并在預(yù)期的細(xì)胞類型中測(cè)試它們的效率。
2.5.2 Approaches for sgRNA construction and delivery
2.5.3 Design of repair template--略拿霉，用的時(shí)候自然會(huì)狠狠學(xué)
2.5.4 Clonal isolation of cell lines
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.
使用流式分離或者連續(xù)稀釋法得到單克隆吟秩。
2.5.5 Functional testing--暫略，用的時(shí)候會(huì)詳細(xì)查閱绽淘，這屬于后續(xù)驗(yàn)證的方法
（1）SURVEYOR nuclease assay.
（2）Plasmid- or ssODN-mediated HDR.
（3）Detection of indels or HDR by sequencing
3. material
4. procedure
5. anticipated results
感謝師兄DZ
