1. Guide RNA Functional Modules Direct Cas9 Activity and Orthogonality
Alexandra E. Briner, Paul D. Donohoue, Ahmed A. Gomaa, Kurt Selle, Euan M. Slorach, Christopher H. Nye, Rachel E. Haurwitz, Chase L. Beisel, Andrew P. May, Rodolphe Barrangou 
Molecular Cell 
Volume 56, Issue 2, Pages (October 2014)
DOI: /j.molcel
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2. Molecular Cell 2014 56, 333-339DOI: (10.1016/j.molcel.2014.09.019)
Copyright © 2014 Elsevier Inc. Terms and Conditions

3. Figure 1 sgRNA Functional Modules
(A) Overview and nomenclature of modules for the sgRNA of the SpyCas9. The six modules include the spacer responsible for DNA targeting (black); the upper stem (blue), bulge (orange), and lower stem (green) formed by the CRISPR repeat:tracrRNA duplex and the nexus (red) and hairpins (purple) from the 3′ end of the tracrRNA.
(B) sgRNA guide variants. Only the altered modules are shown, and mutated nucleotides are represented in red.
(C and D) (C) Biochemical and (D) cell-based DNA cleavage assays were performed with each variant in combination with the SpyCas9, as described in the Experimental Procedures and Supplemental Information. Results are representative of at least three independent experiments. Numbers below lanes indicate average percentage of cleavage observed (n = 3). Results for a complete set of variants are shown in Table S1 and Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2 crRNA:tracrRNA Modules Conservation
(A) Phylogenetic tree based on tracrRNA nucleotide sequences from various Streptococcus and Lactobacillus species. The sequences clustered into three families depicted in blue, orange, and green.
(B) Consensus sequence and secondary structure of the predicted crRNA:tracrRNA for each family. Each consensus RNA is composed of the crRNA (left) base-paired with the tracrRNA. Fully conserved bases are in color, variable bases are in black (two possible bases) or represented by black dots (at least three possible bases), and base positions not always present are circled. Circles between positions indicate base pairing present in only some family members.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 3
The Nexus and Hairpins Dictate Orthogonality between sgRNAs and Cas9
(A) sgRNA sequences for Sth3Cas9 (top, blue) and the Sth1Cas9 (bottom, orange). Both systems are from Streptococcus thermophilus.
(B) Protospacer-targeting scheme. The PAM for each sgRNA is shown. Triangles designate the putative cut sites for each Cas9.
(C) Cas9:chimeric-sgRNA orthogonality in E. coli. Each sgRNA (outside) was subjected to the transformation assay (inside) in E. coli expressing the Sth3Cas9 (blue) or the Sth1Cas9 (orange). Low transformation efficiencies indicate functional Cas9:sgRNA pairs through lethal self-targeting of the E. coli genome. Values reflect the geometric mean and SEM of three independent experiments.
Molecular Cell  , DOI: ( /j.molcel )
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