...permanently modifying the genome dCas9-repressor (such as dCas9-KRAB) or dCas9 gRNA(s) targeting promoter elements... elements of target gene dCas9-KRAB is more effective than dCas9 alone for mammalian cell lines. Activate...without permanently modifying the genome dCas9-activator (such as dCas9-VP64) gRNA(s) targeting promoter elements...select a target sequence and design your gRNA. Know your cell line/organism and genomic sequence When possible...should sequence the region you are planning to modify prior to designing your gRNA, as sequence variation...activate or repress a target gene using dCas9-activators or dCas9-repressors, gRNAs should be targeted to...identifying all PAM sequences within the genetic region to be targeted. If there are no PAM sequences for your chosen...

...achieved by using orthogonal dCas9s (e.g. S. pyogenes dCas9 and S. aureus dCas9) tagged with different fluorescent... be any ~20 nucleotide sequence, provided it meets two conditions: The sequence is unique compared to ...(PAM) The PAM sequence (NGG) serves as a binding signal for Cas9, but the exact sequence depends on which... target, the seed sequence (8–10 bases at the 3′ end of the gRNA targeting sequence) will begin to anneal...matters. Mismatches between the target sequence in the 3′ seed sequence inhibit target cleavage, while mismatches...determined by both the gRNA sequence and the Cas9 enzyme. Ideally, a gRNA targeting sequence will have perfect ... genetic edits. The dCas9 molecule binds to target DNA based on the gRNA sequence without cleavage. This...