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CRISPR-dCas9系統(tǒng)
dCas9蛋白是Cas9蛋白的突變體,即Cas9內(nèi)切酶的RuvC1和 HNH兩個核酸酶活性區(qū)域同時發(fā)生突變。因此,dCas9蛋白的內(nèi)切酶活性全部消失,只保留由gRNA引導(dǎo)進(jìn)入基因組的能力。
CRISPR-dCas9系統(tǒng)提供了一個研究定點(diǎn)轉(zhuǎn)錄調(diào)控的平臺。在這個平臺中,dCas9主要是與其他效應(yīng)蛋白融合(如GFP、轉(zhuǎn)錄因子、組蛋白修飾等),進(jìn)行基因調(diào)控,基因組成像,染色質(zhì)或DNA修飾以及染色質(zhì)免疫沉淀等。
Figure 8 : The Cas9 null mutant has lost both of its DNA cleavage domains while still retaining its ability to target genomic loci through gRNA:DNA interactions. By fusing effector domains to the Cas9 null mutant, it is possible to activate gene expression (i.e. V964 or NF-kB), repress gene expression (i.e. KRBA domain), visualize genomic loci (i.e. EGFP), perform chromatin remodelling (i.e. TET proteins), and simplify chromatin immunopercipitation (through an antibody epitope tag)