Fig 4. Characterization of single promoter, single transcript MoSAIC designs.

(A) Schematic representation of gRNA lentivector backbone and gRNA transcript designs shown in complex with tracrRNA. MV3.2 contains only the first 12 bp of the 35 bp direct repeat sequence suggesting RNA cleavage is not necessary for Cas9 function. MV7.2 includes a spacer sequence TCCCCGGG (rationally designed to prevent hairpin formation) between gRNA sequences. gRNA1 is the eGFP gRNA_1 and gRNA 2 is eGFP gRNA_2. (B) Sequence of eGFP that was targeted using gRNA1 (red) and gRNA2 (blue). (C) Positional eGFP knockout efficiency was determined by flow cytometric analysis. All data shown are averages from three independent experiments. Error-bars represent standard-errors of mean (n = 3).