Genome engineering using the CRISPR-Cas9 system
最近要開(kāi)始學(xué)習(xí)CRISPR-Cas9實(shí)驗(yàn)，對(duì)著動(dòng)輒幾十頁(yè)的說(shuō)明，實(shí)在是看不下去，不如就嘗試用讀書(shū)筆記的方式來(lái)學(xué)習(xí)吧。
今日要講的當(dāng)然是張峰老師組的protocol
文章結(jié)構(gòu)簡(jiǎn)單整理如下：
- abstract
- introduction
- material
- procedure
- anticipated results
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常見(jiàn)縮寫(xiě)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)。
- DNA double-stranded breaks (DSBs) ：雙鏈斷裂，啊啊啊啊好疼?。?/li>
- nonhomologous end joining (NHEJ)：無(wú)模板修復(fù)，引入indel，適合敲除
- homology-directed repair (HDR)：有模板修復(fù)，精準(zhǔn)編輯。
- 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
1.1 Targeted nucleases are powerful tools for mediating genome alteration with high precision. 照例說(shuō)很需要強(qiáng)有力的基因編輯工具。
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. 簡(jiǎn)單說(shuō)由gRNA引導(dǎo)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. 非同源連接(NHEJ)或同源定向修復(fù)(HDR)
1.4 To minimize off-target cleavage, we further describe a double-nicking strategy using the Cas9 nickase mutant with paired guide RNAs. 雙切
This protocol provides experimentally derived guidelines for the selection of target sites, evaluation of cleavage efficiency and analysis of off-target activity. 本protocol提供了選擇靶點(diǎn)的策略、評(píng)價(jià)切割的有效性和脫靶效應(yīng)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. 從靶點(diǎn)設(shè)計(jì)開(kāi)始，基因修飾可在1-2周內(nèi)完成，而2-3周內(nèi)可得到克隆細(xì)胞系。
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2. introduction（節(jié)選）
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.
近年來(lái)出現(xiàn)的基因編輯技術(shù)：ZFNs（鋅指核酸酶），TALENs（轉(zhuǎn)錄激活因子樣效應(yīng)物核酸酶），CRISPR-Cas核酸酶系統(tǒng)。
The first two technologies use a strategy of tethering endonuclease catalytic domains（連接內(nèi)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 （在引導(dǎo)RNA的幫助下）through Watson-Crick base pairing with target DNA
2.1 Precise genome editing using engineered nucleases--精確基因編輯
和ZFN，TALEN一樣，CRISPR-Cas也是通過(guò)激活DSB的模式來(lái)達(dá)到基因標(biāo)記的目的。
在CRISPR-Cas系統(tǒng)中，經(jīng)Cas剪切形成DSB后，DNA可通過(guò)以下兩種途徑進(jìn)行修復(fù)：（A）在缺乏修復(fù)模板的情況下，DSBs通過(guò)NHEJ過(guò)程重新連接，以插入/刪除(indel)突變的形式留下疤痕，可用于基因敲除，indel的出現(xiàn)導(dǎo)致移碼突變和終止密碼子的過(guò)早出現(xiàn)。（B）在DNA修復(fù)模板的情況下，精確修復(fù)-可達(dá)到精確編輯的效果；修復(fù)模板可以是插入序列兩側(cè)帶有同源臂的傳統(tǒng)雙鏈DNA靶向結(jié)構(gòu)，也可以是單鏈DNA寡核苷酸(ssODNs)。
以下這句話很重要：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--詳細(xì)介紹Cas9
簡(jiǎn)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是細(xì)菌用來(lái)抵抗外來(lái)生物抵御系統(tǒng)。經(jīng)過(guò)廣譜檢測(cè)，人們發(fā)現(xiàn)了三種主要的CRISPR系統(tǒng)，它們由CRISPR-associated (Cas)基因、非編碼rna和一組獨(dú)特的重復(fù)元素(直接重復(fù))組成，而這些重復(fù)序列則由來(lái)自外源性DNA靶點(diǎn)(即原間隔體)的短可變序列直接間隔開(kāi)來(lái)；重復(fù)序列+間隔序列=CRISPR RNA (crRNA) array。在有DNA靶點(diǎn)的情況下，每一個(gè)間隔序列都有一個(gè)前間區(qū)序列鄰近基序（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系統(tǒng)是最具特征的系統(tǒng)之一，它由核酸酶Cas9、編碼引導(dǎo)rna的crRNA陣列和有助于將crRNA陣列加工成離散單元的所需輔助反式激活crRNA (tracrRNA)組成。
Cas9酶+（編碼出gRNA的序列+反式激活crRNA，這兩部分都是crRNA array的結(jié)果）
主要是crRNA有什么用，為什么要形成離散單元？
CRISPR RNA (crRNA) array，編碼gRNA，再加上tracrRNA，則可達(dá)到定位+編輯的功能gRNA用于引導(dǎo)，tracrRNA用于結(jié)合靶點(diǎn)。
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，人們就把crRNA和tracrRNA合在一起，成為了single-guide RNA，即sgRNA，而通過(guò)修改tracrRNA的序列，在理論上可以on-target任何目的靶點(diǎn)。
目前應(yīng)用的經(jīng)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 （獲取基因工程鼠的好幫手?。?br>
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在這里，我們?cè)敿?xì)解釋了如何使用人類密碼優(yōu)化，核定位序列兩側(cè)野生型(WT) Cas9核酸酶或突變Cas9核酸酶促進(jìn)真核細(xì)胞基因編輯。
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
3. material
4. procedure
5. anticipated results
感謝師兄DZ
