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1、旗开得胜 读万卷书 行万里路 1 【代码 M0803】SAM target sgRNA cloning protocol- CRISPR-Cas9 实验原理 除基因组编辑技术之外，CRISPR 系统可以被改造成内源基因表达上调的系统。通过修饰 CRISPR 系统中的一些元件，形成协同激活介质（SAM）复合物，成为一种高效特异地转 录激活系统。SAM 复合物中的一个元件是修饰过的 Cas9 核酸酶。SAM 系统中，灭活 Cas9 的催化域形成 dCas9 后与转录激活域（vp64）进行融合。经靶特异向导 RNA (sgRNA)引导，dCas9-VP64-sgRNA 复合物靶向内源基因转录起始位点
2、 （Transcriptional Start Site (TSS)）的-200bp 处，上调基因表达。 SAM target sgRNA cloning protocol S.Konermann, Zhang lab, 2014 Optimized sgRNAs for any coding human genecan be found using our SAM Cas9 activator design tool: :sam.genome-engineering.org/database/ In order to clone the guide target sequenceinto t
3、he sgRNA(MS2) cloning backbone (addgene #61424 ) or lenti sgRNA(MS2)_zeobackbone (addgene #61427), synthesize two oligos of the following form. Bothplasmids have the same overhangs: 旗开得胜 读万卷书 行万里路 2 Example oligo design: Note that the NGG PAM is not included in the designed oligos. Oligonucleotide o
4、rdering tips: Standard de-salted oligos (usually the mostinexpensive synthesis) are sufficient for cloning. If not already resuspended,dilute each oligo to 100M in sterile water or TE. Golden-Gate sgRNA cloning protocol 1. Oligo anneal Mix the components aboveand anneal in a thermal cycler with the
5、following conditions: 37Cfor 30min 95Cfor 5 min Rampto 25C at 5C/min 2. Golden Gate reaction 旗开得胜 读万卷书 行万里路 3 Add90ul of PCR clean H2O to the finished oligoanneal from above to dilute it 1:10. Then mix the following components: Note: use BbsI enzyme for the non-lentiviral SAM sgRNA backbone(addgene
6、#61424) and BsmBI enzyme for the lenti SAM sgRNA (zeo) backbone (addgene#61427). Runthe following program on a thermal cycler: 37Cfor 5 min 20Cfor 5 min repeatfor 15 cycles total Transform2ul of the golden gate reaction in Stbl3 (or other recombination deficient) competentcells. Plate onto Ampicilli
7、n plates. In general, picking 2-3 colonies per guides shouldbe sufficient to ensure a correct clone. Note: it is not necessary to perform a negative control golden-gate reaction(without insert) as it will always contain colonies and is not a good indicatorof cloning success. 更多分子生物学实验方法请点击下方“阅读原文”或者回复“M0001”。 旗开得胜 读万卷书 行万里路 4
