Improved CRISPR/Cas9 knock-in efficiency via the self-excising cassette (SEC) selection method in C. elegans.
Improved CRISPR/Cas9 knock-in efficiency via the self-excising cassette (SEC) selection method in C. elegans.
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DOI：
10.17912/micropub.biology.000460
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发表时间：
2021
影响因子：
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通讯作者：
Doonan R
中科院分区：
文献类型：
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作者：
Huang G;de Jesus B;Koh A;Blanco S;Rettmann A;DeMott E;Sylvester M;Ren C;Meng C;Waterland S;Rhodes A;Alicea P;Flynn A;Dickinson DJ;Doonan R
针对C.六年前首次引入了秀丽隐杆线虫（Dickinson et al. 2015; Schwartz and Jorgensen 2016）。虽然这些基于选择的方法是强大的，但一个缺点是需要注射大量的P0蠕虫（每个基因靶标约30-60个）。我们发现，高纯度DNA和较低浓度的Cas9/sgRNA质粒的组合显著提高了效率，通常通过少至10个注射的蠕虫产生多个独立的CRISPR敲入，与熔化的dsDNA模板和纯化的Cas9蛋白的效率相当（Dokshin et al. 2018; Ghanta and Mello 2020）。
Streamlined, selection-based CRISPR knock-in protocols for C. elegans were first introduced six years ago (Dickinson et al. 2015; Schwartz and Jorgensen 2016). Though these selection-based approaches are powerful, one drawback has been the requirement to inject large numbers of P0 worms (~30-60 per gene target). We have found that a combination of high-purity DNA and a lower concentration of Cas9/sgRNA plasmid dramatically improves efficiency, often resulting in multiple independent CRISPR knock-ins via as few as 10 injected worms, comparable to the efficiency of melted dsDNA templates and purified Cas9 protein (Dokshin et al. 2018; Ghanta and Mello 2020).