ONT Crispr-Cas9 enriched shattering-associated region of P. vulgaris L. Midas
CrRNAs were divided into two pools so that Pool1 would excise subregions 1, 3 and 5 and Pool2 would excise subregions 2 and 4. Two separate mixtures of 6 (Pool 1) and 4 (Pool 2) crRNAs were prepared, and each mixture was completed with transactivation crRNA and duplex buffer and then denatured and cooled to room temperature. RNPs 1 and 2 were formed by mixing each gRNA pool with HiFi Cas9 Nuclease V3 in CutSmart Buffer and incubating at RT. After genomic DNA cutting was completed, the two reactions were pooled together, and the mixture was purified using AMPure XP magnetic beads. Beads were washed twice using Long Fragment Buffer to remove short fragments and DNA was eluted using Elution Buffer.