CRISPR enrichment of spinach 16rRNA barcode
We enriched target chloroplast regions using the nanopore Cas9-targeted sequencing method (nCATS; Gilpatrick et al., 2020). We assembled each RNP complex by adding 1 _l of pooled crRNAs and 1 _l of Alt-R CRISPR-Cas9 tracrRNA to 8 _l of nuclease free water and incubating at 95C for 5 minutes. The RNP complex was then created by incubating 1.2 _l Alt-R HiFi Cas9 Nuclease, 2.8 _l 10X CutSmart Buffer, 23 _l nuclease free water and 3 _l of the gRNA duplex at room temperature for 20 minutes. To selectively enrich the region of interest, we first dephosphorylated pre-existing DNA ends before cutting with Cas9 to preferentially ligate sequencing adapters to the cut sites created by the RNP complex. We did this by incubating 1.5 ng of genomic DNA, 3 _l 10X CutSmart buffer, and 3 _l QuickCIP enzyme at 37C for 10 minutes, followed by enzyme inactivation at 80C for 2 minutes. Cleavage and dA-tailing of the dephosphorylated DNA occurred in a reaction using 10 _l of the assembled RNP complex, 10 mM dATP, and 1 _l Taq DNA polymerase with incubation at 37C for 15 minutes followed by 72C for 5 minutes. Nanopore sequencing adapters were ligated to Cas9 cut sites by incubating 10 _l of NEBNext Quick T4 DNA Ligase, 20 _l ligation buffer, 4.5 _l nuclease free water, and 3.5 _l AMX sequencing adapters at room temperature for 10 minutes. An equivolume amount of TE buffer was then added to the ligated sample, followed by 0.3X volume addition of AMPure XP beads. The library was purified twice using 200 _l long-fragment buffer. We eluted the ligated sample by adding 15 _l elution buffer and incubating at room temperature for 30 minutes before separating the eluate from beads on the magnetic rack. The resulting libraries were sequenced on a MinION Mk1B nanopore sequencer (ONT) using FLO-MIN106D (R9.4.1) flow cells, MinKNOW software (version 22.08.9; ONT), and fast basecalling using Guppy 6.2.11.