The human codon-optimized Cas9 gene [2 (link)] was obtained from Addgene (plasmid 41815). Two flanking primers with added NheI and SacII sites were used to amplify the coding sequence, including the SV40 nuclear localization signal, with the KAPA HiFi polymerase (KAPA BioSystems). The amplicon was digested with the two restriction enzymes and ligated to the vector, pM35S, between the double-enhancer 35S promoter and nopaline synthase (nos) terminator (Additional file 6 ). The entire cassette is flanked with I-SceI restriction sites, which were used to move the Cas9 cassette into p201N to create p201N:Cas9 (Addgene plasmid 59175). The p201N vector is a p201BK [37 (link)] vector modified to include an nptII selectable marker cassette and I-SceI and I-PpoI restriction sites (Additional file 6 ).
For biolistic transformation of soybean, a pSMART HC Kan (Lucigen Corporation, [GenBank: AF532107]) cloning vector was modified to contain a hygromycin phosphotransferase (hph) gene under the control of the Solanum tuberosum Ubi3 promoter and terminator [38 (link)] and the meganuclease I-PpoI site, and is referred to as pSPH2. The vector pSPH2 was digested with I-PpoI and DNA overhangs were removed with T4 DNA polymerase. To prepare the Cas9 insert, p201N:Cas9:gRNA-Glyma07g14530 was digested with SpeI and PmeI and DNA overhangs were removed with T4 DNA polymerase. The vector and insert were ligated to create the plasmid pSPH2:Cas9:gRNA-Glyma07g14530. The Glyma07g14530 gRNA was then replaced with the 01g + 11gDDM1 (Glyma01g38150 and Glyma11g07220) gRNA via I-PpoI to produce pSPH2:Cas9:gRNA-01g + 11gDDM1.
Additional binary Cas9 vectors were produced by replacing nptII from p201NCas9, with hph, bar (phosphinothricin resistance), or GFP. The hph cassette was moved from pSPH2 into the p201N Cas9 vector with the PacI and SpeI restriction sites to produce p201H:Cas9 (Addgene plasmid 59176). The bar and GFP cassettes (double-enhancer 35S promoter, nos terminator) were amplified with the SpeI 35SF and PacI nosR primers (Additional file7 ), and moved into the p201N Cas9 vector with the PacI and SpeI restriction sites to produce p201B:Cas9 (Addgene plasmid 59177) and p201G:Cas9 (Addgene plasmid 59178).
The gRNA targets were designed as previously described [2 (link)], with the exception of the U6 promoter, which was replaced with the Medicago truncatula U6.6 polymerase III promoter [39 (link)] for efficient transcription in soybean. For the gRNA targets, 22-23-bp targets were chosen that had the GN19-20GG motif as previously described [2 (link)]. The GFP 5′- and 3′-targets were chosen because they contain restriction sites that can be used for downstream analysis; however, given the high DNA-modification frequencies, such analyses were not performed. The GN18-19 portion of the genomic target motif was incorporated into the gRNA target molecule. The GFP, Glyma07g14530, and DDM1 gRNA target sequences were synthesized by IDT using gBlocks. The gBlocks were amplified by PCR with flanking primers containing I-PpoI restriction sites. All primer sequences can be found in Additional file7 . The products were then digested with I-PpoI and inserted into the p201N vector. The MET1 (Glyma04g36150 and Glyma06g18790), miR1514, and miR1509 gRNA target sequences were produced with the pUC gRNA shuttle vector system described below. Plasmids were electroporated into Agrobacterium rhizogenes strain K599 and used for hairy-root transformation. Vectors containing both the Cas9 and gRNA target cassettes were combined by inserting the gRNA target cassette into the p201N Cas9 I-PpoI site.
For biolistic transformation of soybean, a pSMART HC Kan (Lucigen Corporation, [GenBank: AF532107]) cloning vector was modified to contain a hygromycin phosphotransferase (hph) gene under the control of the Solanum tuberosum Ubi3 promoter and terminator [38 (link)] and the meganuclease I-PpoI site, and is referred to as pSPH2. The vector pSPH2 was digested with I-PpoI and DNA overhangs were removed with T4 DNA polymerase. To prepare the Cas9 insert, p201N:Cas9:gRNA-Glyma07g14530 was digested with SpeI and PmeI and DNA overhangs were removed with T4 DNA polymerase. The vector and insert were ligated to create the plasmid pSPH2:Cas9:gRNA-Glyma07g14530. The Glyma07g14530 gRNA was then replaced with the 01g + 11gDDM1 (Glyma01g38150 and Glyma11g07220) gRNA via I-PpoI to produce pSPH2:Cas9:gRNA-01g + 11gDDM1.
Additional binary Cas9 vectors were produced by replacing nptII from p201NCas9, with hph, bar (phosphinothricin resistance), or GFP. The hph cassette was moved from pSPH2 into the p201N Cas9 vector with the PacI and SpeI restriction sites to produce p201H:Cas9 (Addgene plasmid 59176). The bar and GFP cassettes (double-enhancer 35S promoter, nos terminator) were amplified with the SpeI 35SF and PacI nosR primers (Additional file
The gRNA targets were designed as previously described [2 (link)], with the exception of the U6 promoter, which was replaced with the Medicago truncatula U6.6 polymerase III promoter [39 (link)] for efficient transcription in soybean. For the gRNA targets, 22-23-bp targets were chosen that had the GN19-20GG motif as previously described [2 (link)]. The GFP 5′- and 3′-targets were chosen because they contain restriction sites that can be used for downstream analysis; however, given the high DNA-modification frequencies, such analyses were not performed. The GN18-19 portion of the genomic target motif was incorporated into the gRNA target molecule. The GFP, Glyma07g14530, and DDM1 gRNA target sequences were synthesized by IDT using gBlocks. The gBlocks were amplified by PCR with flanking primers containing I-PpoI restriction sites. All primer sequences can be found in Additional file
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