Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system
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"Programmable repression and activat..." refers background in this paper
...Mutations D10A and H840A in the RuvC and HNH domains, respectively, abolish cleavage but do not impair DNA binding (8)....
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...In contrast, efficient in vitro cleavage by Cas9 requires 15 nt of homology (5 mismatches) (8)....
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...Cas9 is a crRNAguided double-stranded DNA endonuclease with two domains, RuvC and HNH, each of which cleaves one strand within the target DNA (7,8)....
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...The region upstream of the promoter was changed to include multiple NGG PAM sequences on both strands....
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...To facilitate measurements, we used a GFP reporter plasmid, pWJ89, with the gfp-mut2 gene under the control of a weak biobrick promoter (BBa_J23117) that is preceded by a sequence rich in NGG PAM sequences on both strands....
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"Programmable repression and activat..." refers methods in this paper
...Plasmid pdCas9 was constructed by introducing D10A and H840A mutations to cas9 on pCas9 through amplification of this plasmid with primers B337/B340 and B338/ B339, followed by Gibson assembly (34) of the two products....
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