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/).