High-Throughput Specificity Profiling of crRNAs

High-throughput specificity profiling of unmodified, BNA^NC-modified and LNA-modified crRNAs was performed as previously described^{40 (link)}. Briefly, 200 nM of concatemeric pre-selection libraries were incubated with 1000 nM Cas9 and 1000 nM gRNA or 100 nM Cas9 and 100 nM gRNA in Cas9 cleavage buffer (NEB) for 20 min at 37 °C. Pre-selection libraries were also separately incubated with 2 U of BspMI (NEB) in NEBuffer 3.1 for 1 h at 37 °C. Cas9-digested and BspMI-digested library members were purified with the QiaQuick PCR Purification Kit (Qiagen) and ligated to 10 pmol adaptor1/2(#) (post-selection) or lib adapter 1/lib adapter 2 (pre-selection) (sequences in Supplementary Table 9) with 1000 U of T4 DNA Ligase (NEB) in NEB T4 DNA Ligase Reaction Buffer for 16 h at room temperature. Adapter ligated DNA was purified using the QiaQuick PCR Purification Kit (Qiagen) and PCR amplified for 19–24 cycles with Q5 Hot Start High-Fidelity DNA Polymerase (NEB) in Q5 Reaction Buffer using primers PE2 short/sel PCR (post-selection) or primers lib seq PCR/lib fwd PCR (pre-selection) (sequences in Supplementary Table 9). PCR products were gel purified and quantified using a Qubit 2.0 Fluorometer (ThermoFisher) and subject to single-read sequencing on an Illumina MiSeq. Pre-selection and post-selection sequencing data were analyzed as previously described^{40 (link)}.