Extended Data Figure 7. Predicted structures of guide RNA and purification schema for in vitro biochemistry studies.

a, The CRISPR repeat and tracrRNA anti-repeat are depicted in black whereas the spacer-derived sequence is shown as a series of green N’s. No clear termination signal can be predicted from the locus, so three different tracrRNA lengths were tested based on their secondary structure – 69, 104, and 179 nt in red, blue, and pink, respectively. b, Engineered single-guide RNA corresponding to dual-guide in (a). c, Dual-guide for ARMAN-4 Cas9 with two different hairpins on 3′ end of tracrRNA (75 and 122 nt). d, Engineered single-guide RNA corresponding to dual-guide in (c). e, Conditions tested in E. coli in vivo targeting assay. f, ARMAN-1 (AR1) and ARMAN-4 (AR4) Cas9 were expressed and purified under a variety of conditions as outlined in the Methods section. Proteins outlined in blue boxes were tested for cleavage activity in vitro. g, Fractions of AR1-Cas9 and AR4-Cas9 purifications were separated on a 10% SDS-PAGE gel.