title： Biased genome editing using the local accumulation of DSB repair molecules system
DOI: 10.1038/s41467-018-05773-6
名詞學(xué)習(xí)
microhomology-mediated end-joining (MMEJ)
DNA double-strand break (DSB)
local accumulation of DSB repair molecules (LoAD) system
homologous recombination (HR)
non-homologous end-joining (NHEJ)
homology-independent targeted integration (HITI) system
precise integration into target chromosome (PITCh) system
single-strand template repair (SSTR)
Background and Gaps
Gaps: 以往的研究從未在多個(gè)基因組位點(diǎn)同時(shí)產(chǎn)生多種模式或多個(gè)報(bào)告基因的組合?；虿迦朐诿總€(gè)位點(diǎn)獨(dú)立進(jìn)行，在不同的基因位點(diǎn)上進(jìn)行雙或三重敲入需要一定的步驟。
橫向比較：CRISPR-Cas9基因標(biāo)記使用的方法有（1）同源修復(fù)HR，（2）非同源end-joining NHEJ，（3）微同源介導(dǎo)的end-joining MMEJ。雖然HR的方法可以非常精準(zhǔn)的knockin，但它的載體的構(gòu)建和效率遠(yuǎn)低于end-joining的方法。
Gaps: The NHEJ-mediated homology-independent targeted integration (HITI) system is especially useful for in vivo gene knock-in with high ef?ciency. however, this system cannot assign multiple donors with different genomic loci simultaneously because there are no homology arms on the targeting donor vector for the HITI system.
To overcome these shortcomings, the MMEJ-mediated precise integration into target chromosome (PITCh) system is expected to facilitate high-throughput, simultaneous generation of multiplex knock-in cell libraries because it utilizes short but distinguishable microhomologies (≤40 bp) and results in superior knock-in ef?ciency compared with the HR-mediated method.
工作簡介：
Results
1. Systems development of biased genome editing.
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1.1 以上結(jié)果表明MS2-CtIP LoADing可顯著提高knockin的效率
原因在于：MS2可以與RNA結(jié)合，從而報(bào)告RNA的情況，將MS2與CtIP融合，可將CtIP導(dǎo)向到sgRNA所在的位置，形成一個(gè)滯留，發(fā)揮CtIP增加MMEJ的功效，從而造成了更多的DNA斷裂，插入效率增加。
1.2 knockin效率是增加了，可是編輯的細(xì)胞是雜合子還是純合子？
The results indicated that all three clones obtained with the conventional PITCh system were heterozygous, whereas in two out of eight clones obtained with the LoADed PITCh system, no non-knock-in amplicons were observed, although one of these clones showed another longer amplicon, possibly carrying the plasmid backbone as well as the intended knock-in insert (Supplementary Fig. 7b). These results suggest that homozygous knock-in cells would be established using the LoADed PITCh system even in aneuploid cells, such as HEK293T
LoADed PITCh system能得到純合子的概率是2/8，2個(gè)純合子中有一個(gè)longer amplicon，而單純的PITCh system全部都得到雜合子。
1.3 knockin效率增加了，還能得到純合子，那么脫靶情況如何？
No evidence of off-target integrants was detected among eight clones, except that one minor band showing slightly longer size than expected was observed in one out of eight clones
1.4 會(huì)有細(xì)胞毒性嗎？
之前我看過一篇文章，說的是CRISPR-Cas9的效果由于P53的存在而大打折扣，而P53對抗HDR是CRISPR-Cas9造成DSB無法被修復(fù)，從而介導(dǎo)了CRISPR-Cas9的細(xì)胞毒性。那么就會(huì)有以下兩種情況：（1）P53存在時(shí)，CRISPR-Cas9效果不好，且有細(xì)胞毒性；（2）P53敲除時(shí)，CRISPR-Cas9效率增加，但有致癌的風(fēng)險(xiǎn)。以此引發(fā)臨床安全性的思考，提醒在人體上使用CRISPR-Cas9治療需要注意的安全性問題，從而以一個(gè)相對簡單的故事，發(fā)表在了Nature Medicine上。所以，我們在使用基因編輯工具的時(shí)候，需要注意一下它的細(xì)胞毒性情況。
（1）FACS結(jié)果顯示，沒有毒性：首先轉(zhuǎn)入已被證實(shí)具有細(xì)胞毒性的載體來作為對照，MS2-CtIP組沒有對細(xì)胞增殖產(chǎn)生影響，而ZFN組有。
（2）如我前面所說，DSB如無法被修復(fù)，則是細(xì)胞毒性。在這里作者也使用DSB修復(fù)實(shí)驗(yàn)來代表細(xì)胞毒性實(shí)驗(yàn)，首先使用依托泊苷誘導(dǎo)DSB，然后使用anti-γ-H2AX染色來查看修復(fù)情況，發(fā)現(xiàn)MS2-CtIP組DSB修復(fù)活性顯著高于對照組。
以上結(jié)果表明，MS2-CtIP是通過誘導(dǎo)DSB修復(fù)來達(dá)到低（無）細(xì)胞毒性的效果。
1.5 一般情況下，還會(huì)再問一下MS2-CtIP增加knockin效率的機(jī)制
我就不寫了。。。因?yàn)槲覜]看明白，嚶嚶嚶。
2. Characterization of DSB repair pathway choice.
第一部分主要講基因編輯系統(tǒng)的構(gòu)建及基本情況，接下來就要講一講它作為一個(gè)基因編輯工具的基本素養(yǎng)了。
太多啦！簡單說一下，就是對比了MMEJ和NHEJ它們在精確敲入，非精確敲入和未敲入這三個(gè)方面的情況，得到MMEJ幾乎完敗NHEJ的結(jié)論咯。看看這圖畫得多漂亮！
3. Parallel generation of multiplex knock-in cell collections.
回歸前面說的Gaps，同時(shí)對多個(gè)基因進(jìn)行編輯呢？結(jié)果顯示是可以高效、準(zhǔn)確的對多個(gè)基因進(jìn)行編輯。這部分是灰?；页０舻?。
4. Versatility of the LoAD system.
Finally, we found the common knock-in-enhancing effect of LoADed MS2-CtIP on single-strand template repair and HR repair pathways.
總結(jié)
學(xué)習(xí)一下人家的思路~
創(chuàng)新點(diǎn)在于MS2的定位效應(yīng)，MS2-CtIP的增強(qiáng)效應(yīng)，MMEJ的精巧性。
參考文獻(xiàn)： Nakade S, Mochida K, Kunii A, et al. Biased genome editing using the local accumulation of DSB repair molecules system[J]. Nature communications, 2018, 9(1): 3270.
