Rationally Designed Anti-CRISPR Nucleic Acid Inhibitors of CRISPR-Cas9

SCHEME 1.. Chemical modifications and nucleic acid-based inhibitor modification schemes used in this study. LNA, locked nucleic acid; PAM, protospacer adjacent motif; PEG, polyethylene glycol; PS, phosphorothioate.

FIG. 1.. Nucleic acid-based inhibitor module design and activity. (A) Points of contact for nucleic acid-based inhibitors of CRISPR-SpCas9 [33]. SpCas9 protein is depicted in brown, tracrRNA in blue, crRNA in green, complementary DNA target in red, and PAM-containing DNA in yellow. (B) Illustration of a dgRNA for CRISPR-SpCas9. sgRNA (not shown) consists of a shorter crRNA-tracrRNA pairing stem that is closed by a GNRA tetraloop [2]. (C–E) Sequence, secondary structure, and pairing of nucleic acid-based inhibitor modules. (F) Binding curves and calculated affinity of inhibitor modules for the SpCas9-tracrRNA or SpCas9-tracrRNA-crRNA RNP complexes determined by dot-blot filter binding. Error bars are standard error of the mean (SEM). CRISPR, clustered regularly interspaced short palindromic repeat; dgRNA, dual-guide RNA; PAM, protospacer adjacent motif; PS, phosphorothioate; RNP, ribonucleoprotein; SEM, standard error of the mean; sgRNA, single-guide RNA.

FIG. 2.. Modular nucleic acid-based inhibitors and their binding to SpCas9-tracrRNA RNP complex. Sequence, secondary structure, and pairing for each inhibitor design are shown in the left panels (A, C, E, and G) and the associated binding activity and calculated affinity are shown in the right panels (B, D, F, and H). Error bars are standard error of the mean (SEM).

FIG. 3.. Inhibition of CRISPR-SpCas9 catalytic activity in vitro with nucleic acid-based inhibitors. (A) Inhibition of in vitro SpCas9 cleavage activity over time. Inhibitor and crRNA (crE2) concentrations were set at 1 μM. Error bars are standard error of the mean (SEM). (B) Inhibition of 10 min in vitro cleavage assay reactions containing 0.25 μM tracrRNA and 0.3 μM crRNA (crEGIP). Error bars are standard error of the mean (SEM). (C) Inhibition of the same reaction, but with 0.25 μM sgRNA (sgEGIP). Error bars are standard error of the mean (SEM).

FIG. 4.. Inhibition of CRISPR-SpCas9 editing in human cells by nucleic acid-based inhibitors. (A) Inhibition of cell-based editing by lipid co-transfection of guide RNAs and inhibitors at a 1:1 molar ratio into HEK 293T cells expressing EGFP and SpCas9. Editing was measured by flow cytometry. Error bars are standard error of the mean (SEM). (B) Dose-dependent inhibition of editing in human cells with sgRNA and inhibitors. sgRNA and inhibitor were lipid co-transfected into HEK 293T cells expressing EGFP and SpCas9 or sgRNA-Cas9 RNP was co-electroporated into HEK 293T cells expressing EGFP. Error bars are standard error of the mean (SEM). EGFP, enhanced green fluorescent protein.