Single Guide RNA Library Design and Construction.
Single Guide RNA Library Design and Construction.
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DOI：
10.1101/pdb.prot090803
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发表时间：
2016-03-01
影响因子：
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通讯作者：
Sabatini DM
中科院分区：
文献类型：
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作者：
Wang T;Lander ES;Sabatini DM
该方案描述了如何生成用于遗传筛选的单向导RNA（sgRNA）文库。有许多在线工具可用于预测具有高靶特异性和/或切割活性的sgRNA序列。在这里，我们向用户推荐我们为人类和小鼠开发的全基因组sgRNA序列预测，这些预测可从Broad Institute网站获得。一旦一组靶基因和相应的sgRNA序列被鉴定，定制的寡核苷酸池可以由许多商业供应商快速合成。此后，如本文所述，可以将寡核苷酸有效地克隆到适当的慢病毒表达载体骨架中。然后可以将所得质粒库包装到慢病毒颗粒中，并用于在任何选择的细胞系中产生敲除。
This protocol describes how to generate a single guide RNA (sgRNA) library for use in genetic screens. There are many online tools available for predicting sgRNA sequences with high target specificity and/or cleavage activity. Here, we refer the user to genome-wide sgRNA sequence predictions that we have developed for both the human and mouse and that are available from the Broad Institute website. Once a set of target genes and corresponding sgRNA sequences has been identified, customized oligonucleotide pools can be rapidly synthesized by a number of commercial vendors. Thereafter, as described here, the oligonucleotides can be efficiently cloned into an appropriate lentiviral expression vector backbone. The resulting plasmid pool can then be packaged into lentiviral particles and used to generate knockouts in any cell line of choice.