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. Author manuscript; available in PMC: 2024 Aug 17.
Published in final edited form as: Cell Chem Biol. 2023 Jun 29;30(8):879–892.e5. doi: 10.1016/j.chembiol.2023.06.007

Figure 2. TdT-based capture of sgRNA cleavage.

Figure 2.

(A) Variant or WT sgRNAs are complexed to SpCas9 and a radiolabeled or fluorescently labeled DNA substrate. Upon cleavage by Cas9, TdT adds a poly(A) chain to the PAM-distal DNA’s 3’ end. A biotinylated Oligo(dT) probe binds the poly(A) tail, and the whole complex can be captured with magnetic streptavidin coated beads. Captured complexes are analyzed by scintillation counting or flow cytometry. (B) The WT sgRNA was complexed with Cas9 variants and then incubated with a radiolabeled substrate DNA and the components of an A-tailing assay as described in the Methods. As negative and positive controls, the WT sgRNA was complexed with inactive “dead” Cas9 or active Cas9 with (as indicated with ‘(TdT)’) or without TdT. Complexes containing cleaved DNA targets were bound by magnetic streptavidin beads, washed several times, and the amount of labeled DNA in the bead fraction and wash fractions was determined. The DNA only control does not contain either Cas9 or sgRNA and serves as a non-specific bead-binding background control. Error bars represent 4 independent replicates.