Cas9-Mediated Genome Editing Analysis

Plant and bacterial DNA were extracted as previously published [29 (link)]. Forward and reverse primers were designed to anneal to genomic DNA sequences at approximately 250 bp flanking the putative clipping sites (S2 Table). PCR was performed using PfuUltra II Hotstart 2X Master Mix (Agilent Technologies, Inc). After amplification the PCR products were purified using the Zymo Clean and Concentrator Kit (Zymoresearch, USA). For direct sequencing of PCR products, the same primer set for amplifications were used. For sequencing of cloned PCR products, PCR fragments were cloned directly into pCR2.1 using the manufacturer’s recommendations for the Zero Blunt Topo cloning kit (Life Technologies), and sequenced using M13F and M13R primers. In both cases DNA traces were compared to wild type sequences using the SeqMan function from LaserGene analysis package. For directly sequenced PCR products, deviations from a consensus trace that originated near the -3 position relative to the NGG PAM site, the known site of Cas9 cutting, were scored as evidence of genome editing in somatic tissues relative to wild type traces.