Supplementary Figure 6: QCgRNA amplicon expression cassettes.

(a) Schematic showing the various elements of the QCgRNA amplicon primers QCgFwd, QCgX and QCgRev. Sequences annealing to U6 promotor template are shown in orange; gRNA design in red; tracr elements in blue; restriction enzyme sites for sub-cloning to pEPB104 in italics. Note that the gRNA design is incorporated into QCgX as the complementary sequence to the target, which is shown in green. The nucleotide (c) is only included in the QCgX primer, if the gRNA (=target) does not contain a G as the first (5´) nucleotide, which is the case in the present example. (b) Agarose gel (2%) electrophoresis check for the formation of full-length (f) products (arrow) by QCgRNA amplicon tri-primer PCRs, as compared to control (c) PCRs containing only QCgFwd and QCgX primers (Step 3 in Procedure). Various amounts of MassRuler Low Range DNA ladder (M) are run alongside to enable quantitation of the QCgRNA amplicons. (c) The indel profiles elicited by a gRNA design in a QCgRNA amplicon and a plasmid vector are identical, as illustrated by targeting GALNT10 in HEK293 cells. The GALNT10 QCgRNA amplicon was subcloned into pEPB104 plasmid (Addgene #68369; Supplementary Sequence 1) using EcoRI and KpnI restriction endonuclease sites present in both constructs. (d) A QCgRNA design found non-functional in one cell type typically remains non-functional when tested in other cell types, as illustrated with a POMT2 QCgRNA. IDAA profiles were generated in an (c) ABI 3500 or and (d) ABI 3130 instrument.