Genome extraction kit

Genomic DNA was extracted from transgenic tomato leaves using a genome extraction kit (CWBIO, China). PCR amplifications were conducted using primer pairs off-1F and off-1R, off-2F and off-2R, off-3F and off-3R, off-4F, and off-4R for SlGRAS8 CRISPR lines (Table S7). The PCR products were purified by PCR Pure kit (Magen, China). The PCR products were used for the T7 endonuclease I (T7EI, Takara, Japan) assay and monoclonal sequencing analysis. Monoclonal sequencing analysis is briefly described as follows: PCR products were cloned into the pEASY-Blunt vector (Transgene, China). Plasmids from 10 single colonies for each sample were sequenced using the off-1F, off-2F, off-3F, and off-4F. Sequencing was performed by the Tsingke Biotechnologies Company and analyzed manually. The potential off-target mutated sites and their off-target scores were estimated with the CRISPR-P 2.0 design tool (http://cbi.hzau.edu.cn/CRISPR2/).

Genomic DNA from transgenic tomato leaves was extracted using a genome extraction kit (CWBIO, China) to detect mutated sequences. Amplifications of genomic sequence containing the target site were conducted by PCR using the primer pairs provided in Table S6. The PCR products were purified directly using a gel Pure kit (Magen, China). The purified PCR product is directly used for sequencing for T0 plant. Purified PCR products were cloned into the pEASY-Blunt vector (Transgene, China) to observe different types of mutations in T1 plant. Then, monoclonal sequencing was performed using the M13F primer by the Tsingke Biotechnologies Company and analyzed manually.