Wang Tim, Lander Eric S, Sabatini David M
Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139; Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142; Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142; David H. Koch Institute for Integrative Cancer Research at MIT, Cambridge, Massachusetts 02139; Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139;
Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139; Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142; Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115.
Cold Spring Harb Protoc. 2016 Mar 1;2016(3):pdb.prot090803. doi: 10.1101/pdb.prot090803.
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.
本方案描述了如何生成用于基因筛选的单导向RNA（sgRNA）文库。有许多在线工具可用于预测具有高靶向特异性和/或切割活性的sgRNA序列。在此，我们推荐用户使用我们为人类和小鼠开发的全基因组sgRNA序列预测工具，该工具可从布罗德研究所网站获取。一旦确定了一组靶基因和相应的sgRNA序列，许多商业供应商可以快速合成定制的寡核苷酸池。此后，如本文所述，寡核苷酸可以有效地克隆到合适的慢病毒表达载体骨架中。然后可以将所得的质粒池包装成慢病毒颗粒，并用于在任何选择的细胞系中产生基因敲除。
