title： Biased genome editing using the local accumulation of DSB repair molecules system
DOI: 10.1038/s41467-018-05773-6
名词学习
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: 以往的研究从未在多个基因组位点同时产生多种模式或多个报告基因的组合。基因插入在每个位点独立进行，在不同的基因位点上进行双或三重敲入需要一定的步骤。
横向比较：CRISPR-Cas9基因标记使用的方法有（1）同源修复HR，（2）非同源end-joining NHEJ，（3）微同源介导的end-joining MMEJ。虽然HR的方法可以非常精准的knockin，但它的载体的构建和效率远低于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 efficiency. 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 efficiency compared with the HR-mediated method.
工作简介：
Results
1. Systems development of biased genome editing.
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1.1 以上结果表明MS2-CtIP LoADing可显著提高knockin的效率
原因在于：MS2可以与RNA结合，从而报告RNA的情况，将MS2与CtIP融合，可将CtIP导向到sgRNA所在的位置，形成一个滞留，发挥CtIP增加MMEJ的功效，从而造成了更多的DNA断裂，插入效率增加。
1.2 knockin效率是增加了，可是编辑的细胞是杂合子还是纯合子？
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能得到纯合子的概率是2/8，2个纯合子中有一个longer amplicon，而单纯的PITCh system全部都得到杂合子。
1.3 knockin效率增加了，还能得到纯合子，那么脱靶情况如何？
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 会有细胞毒性吗？
之前我看过一篇文章，说的是CRISPR-Cas9的效果由于P53的存在而大打折扣，而P53对抗HDR是CRISPR-Cas9造成DSB无法被修复，从而介导了CRISPR-Cas9的细胞毒性。那么就会有以下两种情况：（1）P53存在时，CRISPR-Cas9效果不好，且有细胞毒性；（2）P53敲除时，CRISPR-Cas9效率增加，但有致癌的风险。以此引发临床安全性的思考，提醒在人体上使用CRISPR-Cas9治疗需要注意的安全性问题，从而以一个相对简单的故事，发表在了Nature Medicine上。所以，我们在使用基因编辑工具的时候，需要注意一下它的细胞毒性情况。
（1）FACS结果显示，没有毒性：首先转入已被证实具有细胞毒性的载体来作为对照，MS2-CtIP组没有对细胞增殖产生影响，而ZFN组有。
（2）如我前面所说，DSB如无法被修复，则是细胞毒性。在这里作者也使用DSB修复实验来代表细胞毒性实验，首先使用依托泊苷诱导DSB，然后使用anti-γ-H2AX染色来查看修复情况，发现MS2-CtIP组DSB修复活性显著高于对照组。
以上结果表明，MS2-CtIP是通过诱导DSB修复来达到低（无）细胞毒性的效果。
1.5 一般情况下，还会再问一下MS2-CtIP增加knockin效率的机制
我就不写了。。。因为我没看明白，嘤嘤嘤。
2. Characterization of DSB repair pathway choice.
第一部分主要讲基因编辑系统的构建及基本情况，接下来就要讲一讲它作为一个基因编辑工具的基本素养了。
太多啦！简单说一下，就是对比了MMEJ和NHEJ它们在精确敲入，非精确敲入和未敲入这三个方面的情况，得到MMEJ几乎完败NHEJ的结论咯。看看这图画得多漂亮！
3. Parallel generation of multiplex knock-in cell collections.
回归前面说的Gaps，同时对多个基因进行编辑呢？结果显示是可以高效、准确的对多个基因进行编辑。这部分是灰常灰常棒的。
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.
总结
学习一下人家的思路~
创新点在于MS2的定位效应，MS2-CtIP的增强效应，MMEJ的精巧性。
参考文献： 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.
