Bsai hf

Multiple sgRNA expression cassettes mixtures (1−2 µg) in an equimolar ratio were digested in a 50-μl reaction with 1 µl of Bsa I-HF (NEB, USA). Digested products were purified using a PCR Pure kit (Magen, China).
Ligation reactions (10 μl) were set up with 1 × T4 DNA ligase buffer, 5 U of T4 DNA ligase (Thermo, USA), 50 ng of Cas9VL, and 20 molar ratios of each digested sgRNA expression cassette (mixture). Reactions were incubated at 22 °C for 1 h. For two to four sgRNA expression cassettes, 10 µl of the ligation mixture was transformed into 50 µl of chemically competent transT1 E. coli (Transgene, China). In cases with five or more sgRNA expression cassettes, 100 µl of chemically competent transT1 E. coli are required. A schematic diagram of the assembly of multiple sgRNA expression cassettes is shown in Fig. 2d.
We prepared eight pairs of Bsa I-site primers; therefore, eight different sgRNA expression cassettes can be assembled by one-step Golden Gate assembly that is compatible with the BioBrick Standard Assembly. Biobricks of four sgRNA expression cassettes can be amplified using Bb-F and Bb-R primers from four ligation products of sgRNA expression cassettes and assembled into Biobricks site of pHNCas9. This strategy is suitable for ligating more than eight sgRNA expression cassettes into pHNCas9. Bb-F and Bb-R primer are provided in Table S2.

The pDR274 vector (Addgene Plasmid 42250), harboring a T7 promoter positioned upstream of a partial guide RNA sequence (Hwang et al., 2013b (link)) was used for sgRNA expression. Appropriately designed oligonucleotides were synthesized with oligonucleotide purification cartridge (OPC) purification at Operon Biotechnologies (Tokyo. Japan). A pair of oligonucleotides (final concentration: 10 µM each) was annealed in 10 µL of annealing buffer (40 mM Tris-HCl [pH 8.0], 20 mM MgCl₂, and 50 mM NaCl) by heating to 95°C for 2 min and then cooling the mixture slowly to 25°C in 1 h. The pDR274 vector was digested with BsaI-HF (New England Biolabs), and the annealed oligonucleotides were ligated into the pDR274 vector. Sequences of the genomic target sites and the annealed oligonucleotides are listed in supplementary material Table S2.

The Golden Gate modular cloning system was used to prepare the plasmids [58 (link)]. This included a restriction digest-ligation protocol of 25 cycles of 3 min at 37°C and 4 min at 16°C using T4 DNA Ligase (New England BioLabs, Ipswich, UK) combined with the restriction enzymes BsaI-HF (New England BioLabs, Ipswich, UK) and BpiI (Thermo Fisher Scientific, Waltham, USA) for level1 and level2 assembly, respectively. All Level 0 s used in this study are held for distribution in the ENSA project core collection (https://www.ensa.ac.uk/) and are listed along with the binary plasmid details in Table S2. Sequences were domesticated, synthesized and cloned into pMS (GeneArt, Thermo Fisher Scientific, Waltham, USA). Sequence information for Medtr7g096530 (LBD16), Medtr4g060950 (LBD11) and Medtr6g086870 (YUC2) and Medtr7g099330 (YUC8) were obtained from the M. truncatula Mt4.0v1 genome via Phytozome (https://phytozome.jgi.doe.gov) [60 (link)]. The Dex-inducible system (GVG and 6xGAL4UAS) was adapted from the original system [59 (link)].