Supplementary Figure 12: Illustration of LmoCascade crRNA cloning scheme for expression in mammalian cells.
From: Targeted transcriptional modulation with type I CRISPR–Cas systems in human cells
(a) Schematic representation of L. monocytogenes Finland_1998 processed crRNA with 5’ PAM recognition and base pairing at the DNA target loci. (b) Engineering of pre-processed crRNA driven by U6 promoter for expression and processing in mammalian cells. (c) Illustration of pAPcrRNA_Lmo cloning plasmid digested with SacII and AgeI. To insert repeat-spacer pairs, forward and reverse oligonucleotides encoding the palindromic repeat and crRNA spacers were synthesized. For each crRNA, the 3’ AgeI site results in mismatched nucleotides between the spacer and target sequences. Annealed oligonucleotide pairs can be annealed, 5’ phosphorylated with PNK and ligated into digested pAPcrRNA_Lmo. Oligo, oligonucleotide.
