Gel pure kit

PEASY–AtMYB75 was digested with Kpn I and Spe I restriction enzyme (Thermo, USA). BioBrick OEAtMYB75 fragments were separated by agarose gel electrophoresis and purified using gel Pure kit (Magen, China). pHNCas9 was digested by Kpn I and Xba I restriction enzyme (Thermo, USA) using PCR pure kit. Ligation reactions (10 μl) were set up with 1 × T4 DNA ligase buffer, 1 U of T4 DNA ligase (Thermo, USA), 50 ng of pHNCas9, and five molar ratios of BioBrick OEAtMYB75. Reactions were incubated at 22 °C for 1 h. A total of 5 µl of the ligation mixture was transformed into 25 µl of chemically competent transT1 E. coli (Transgene, China). The new CRISPR vector containing BioBrick OEAtMYB75 was named pHNCas9:OEAtMYB75 (Fig. 7a).

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.

In the current study, AtU3b-sgRNA, AtU3d-sgRNA, AtU6-26-sgRNA, and AtU6-29-sgRNA mixed templates were used in one-step PCR. Gene-specific primer design for pHNCas9 was adopted using our primer design aid (Excel S1). The gene-specific primer for this protocol was named gene name-AtUX-F in this gadget. In Table S3, we specify the details for using the BsaI site primer. SgRNA expression cassettes were amplified in 50 µl with 0.01 µM gene-specific primer, 0.2 µM each of the corresponding Bsa I-site primer, and 1 µl of corresponding mixed template using TransStart FastPfu Fly DNA Polymerase (Transgene, China) for 45–50 cycles (Fig. 2c). The PCR products were purified using a gel Pure kit (Magen, China). The presumed sequences of sgRNA expression cassettes produced by one-step PCR are provided in Text S3.