Clonexpress 2 one step cloning kit c112

The Arabidopsis thaliana ecotype Col-0, Agrobacterium strain GV3101, Escherichia coli strains TOP10 and DB3.1, and the plant expression vector pMDC162 plasmid used in this study were all provided by our laboratory. TOPO vectors (pENTR™ Directional TOPO^® Cloning Kits) were purchased from Life Technologies, ClonExpress^® II One Step Cloning Kit C112 was purchased from Vazyme, and GUS staining kits were purchased from Solarbio.

All plasmids and guide RNAs used in this study can be found in S1 Table. AsCas12a human expression plasmid pY010 was purchased from the nonprofit plasmid repository Addgene (Addgene plasmids #69982). All AsCas12a variants were generated by standard site-directed mutagenesis. In brief, using AsCas12a plasmid pY010 as the template and a pair of primers that one primer carrying site mutation nucleotide to amplify 500 bp fragments by PCR (Phanta MAX Super-Fidelity DNA Polymerase P505, Vazyme). After confirming that the bands were correct by agarose gel electrophoresis, the PCR products were purified. Next, taking 1,000 ng PCR products as the circling mutation primers and 100 ng AsCas12a plasmid pY010 as template to carry out PCR again. PCR products were purified and use DpnI to digest the original template at 37°C for 1 h, inactivated at 80°C for 20 min, take 10 μl digested products for transformation. The next day, single colonies were sent for sequencing. Oligonucleotide duplexes corresponding to spacer sequences were PCR amplified and cloned into pU6-As-crRNA plasmids (Addgene plasmids #78956) for U6 promoter-driven U6 promoter-driven transcription of As crRNAs (ClonExpress II One Step Cloning Kit C112, Vazyme).

Standards of lycopene (CAS, 502-65-8), β-carotene (CAS, 7235-40-7), zeaxanthin (144-68-3), and crocetin dialdehyde (CAS, 502-70-5) were purchased from Sigma-Aldrich (Sigma-Aldrich Corp., St. Louis, MO, USA). Phanta Max Super-Fidelity DNA Polymerase was purchased from Vazyme (Nanjing Vazyme Biotech Co., Ltd., Nanjing, China). A ClonExpress II One Step Cloning Kit C112 was purchased from Vazyme. IPTG was purchased from Solarbio (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). All restriction endonucleases were purchased from Takara (Takara Biomedical Technology (Beijing) Co., Ltd., Beijing, China). T4 DNA Ligase was purchased from Takara. A DNA plasmid isolation kit and DNA gel extraction kit were purchased from Tiangen (TIANGEN Biotech (Beijing) Co., Ltd., Beijing, China). Tryptone and yeast extract were purchased from Thermo (Thermo Fisher Scientific Inc., Waltham, MA, USA). E. coli DH5α and E. coli BL21 (DE3) were purchased from Tiangen. Z/B/L represent the BL21 (DE3) harboring pACCAR25ΔcrtX, pACCAR16Δcrt, or pACCRT-EIB, respectively. All the chemical reagents utilized in this experiment were of analytical grade.