The vectors were assembled using the Golden Gate modular cloning method [23 (link)]. To generate the Cas9 expression cassettes, the RPS5a, YAO, ICU2, CsVMV, EC1.2, EC_enh., UBI10, AG, MGE1 and 35S promoters, the Cas9_1, Cas9_2, Cas9_3 and Cas9_4 coding sequences, the Ocs, Nos, Ags and E9 terminators were amplified using primers flanked with BpiI restriction sites associated with Golden Gate compatible overhangs (S3 Table ). 0.02 pmoles of the purified PCR products were mixed with the same molar amount of the corresponding Level 0 vector (S3 Table ), 0.5 μl of BpiI enzyme (10U/μl, ThermoFisher), 0.5 μl of T4 ligase (400U/μl, NEB), 1.5 μl of CutSmart Buffer (NEB), 1.5μl of Bovine Serum Albumin (10X) and water in a total reaction volume of 15 μl. The reaction was placed in a thermocycler and the following ‘Golden Gate’ program was applied: 20 seconds 37°C, 25 cycles of [3 minutes 37°C / 4 minutes 16°C], 5 minutes 50°C and 5 minutes 80°C.
Combinations of three Level 0 vectors containing respectively a promoter, a Cas9 coding sequence and a terminator were assembled in Level 1 vector pICH7742 (Position 2) or pICH47811 (Position 2, reverse) by the same ‘Golden Gate’ protocol but using 0.5 μl of BpiI enzyme (10U/μl, ThermoFisher) instead of 0.5 μl of BsaI-HF.
To generate the sgRNA expression cassettes, DNA fragments containing the classic or the ‘EF’ backbone with 7, 67 or 192 bp of the U6-26 terminator were amplified using primers flanked with BsaI restriction sites associated with Golden Gate compatible overhangs (S3 Table ). The amplicons were assembled with the U6-26 promoter (pICSL90002) in Level 1 vector pICH7751 (Position 3) by the ‘Golden Gate’ protocol using the BsaI-HF enzyme. Combinations of three Level 1 vectors containing a glufosinate resistance selectable maker (pICSL11017), a Cas9 expression cassette and a sgRNA expression cassette were assembled in Level 2 pAGM4723 (- overdrive) or pICSL4723 (+ overdrive) by the ‘Golden Gate’ protocol using the BpiI enzyme. All the plasmids were prepared using a QIAPREP SPIN MINIPREP KIT on Escherichia coli DH10B electrocompetent cells selected with appropriate antibiotics and X-gal.
All the plasmid identification numbers refer to the ‘addgene database’ (www.addgene.org/ ).
Combinations of three Level 0 vectors containing respectively a promoter, a Cas9 coding sequence and a terminator were assembled in Level 1 vector pICH7742 (Position 2) or pICH47811 (Position 2, reverse) by the same ‘Golden Gate’ protocol but using 0.5 μl of BpiI enzyme (10U/μl, ThermoFisher) instead of 0.5 μl of BsaI-HF.
To generate the sgRNA expression cassettes, DNA fragments containing the classic or the ‘EF’ backbone with 7, 67 or 192 bp of the U6-26 terminator were amplified using primers flanked with BsaI restriction sites associated with Golden Gate compatible overhangs (
All the plasmid identification numbers refer to the ‘addgene database’ (
Free full text: Click here
