Performance of various hgRNAs. (A) The sequence of seven different hgRNAs. Transcription start site (TSS), spacer sequence, and PAM are marked with grey, blue, and pink boxes, respectively. (B,C,D) Cas9 is induced in cell lines with the hgRNAs from panel A integrated genomically. DNA samples harvested before (t=0 days) and at various points after induction are characterized by high-throughput sequencing to quantify mutations, functionality, and generated diversity for each hgRNA. See also . (E) Design of four longer variants of hgRNA-A21. Stuffer sequences of 5, 30, 55, or 80 base pairs were added upstream of a spacer very similar to the A21 spacer to obtain the four increasingly lengthy A’ variants. (F,G,H) Cas9 is induced in cell lines with the hgRNAs from panel E integrated genomically. DNA samples harvested before (t=0 days) and at various points after induction are characterized by high-throughput sequencing to quantify mutations and functionality of hgRNA loci. Data points represent duplicate experiments.