Fig. 4

Functional analysis of CORALINA gRNAs. a Top: Overview of gRNAs used in experiments to target YFP. gRNA Y1-35, gRNA Y1- 40, gRNA Y2-40 target YFP and contain protospacer with 35 or 40 bp length respectively. Bottom: Flow cytometry reveals that gRNAs with long protospacer (35 or 40 bp) efficiently target the Cas9 protein and induce mutations (detected through YFP loss, transfection rate ca. 30 %). b Top: Schematic depicting a CORALINA derived gRNA targeting the third last intron of the human gene PXDC1 (gRNA P1-44, 44 bp protospacer). gRNA P1-20 is trimmed from the 5′ end to yield a 20 bp protospacer. Below: Bargraph depicting percentage of NGS reads displaying indels resulting from coexpression of wild-type Cas9 with the two gRNAs in HEK293T cells. While NGS sequencing reads of control cells reveal only wildtype sequences, reads derived from cells transfected with gRNA P1-20 or gRNA P1-44 (and S. Pyogenes Cas9) displayed genetic alteration around the targeting sites. Bottom: Microscopy images indicating transfection efficiency is ca. 30 %. c List of the most frequently sequenced alterations generated with gRNA P1-44 and classified as WT (wild-type), INS (insertion), DEL (deletion) or COM (complex). Complex cases relate to cases where more than one insertion or deletion has happened. The sequence highlighted with capital letters correspond to the target of the guide RNA. For insertions and complex events, the ‘>’ ‘<‘mark the location of the event. See Additional file 4: Figure S4A for alterations induced by gRNA P1-20