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MISSA 2.0: an updated synthetic biology toolbox for assembly of orthogonal CRISPR/Cas systems

Efficient generation of plants carrying mutations in multiple genes remains a challenge. Using two or more orthogonal CRISPR/Cas systems can generate plants with multi-gene mutations, but assembly of these systems requires a robust, high-capacity toolkit. Here, we describe MISSA 2.0 (multiple-round in vivo site-specific assembly 2.0), an extensively updated toolkit for assembly of two or more CRISPR/Cas systems. We developed a novel suicide donor vector system based on plasmid RK2, which has much higher cloning capacity than the original, plasmid R6K-based system. We validated the utility of MISSA 2.0 by assembling multiple DNA fragments into the E. coli chromosome, and by creating transgenic Arabidopsis thaliana that constitutively or inducibly overexpress multiple genes. We then demonstrated that the higher cloning capacity of the RK2-derived MISSA 2.0 donor vectors facilitated the assembly of two orthogonal CRISPR/Cas systems including SpCas9 and SaCas9, and thus facilitated the creation of transgenic lines harboring these systems. We anticipate that MISSA 2.0 will enable substantial advancements in multiplex genome editing based on two or more orthogonal CRISPR/Cas9 systems, as well as in plant synthetic biology.


Supplementary Tables
Supplementary Table S1: Please see the Excel file

Supplementary
Supplementary Methods S1

Creation of the donor vectors pLC2-ccdB and pRG2-ccdB
We generated SalI and XhoI-ClaI-flanked oriTF fragment by PCR amplification from pMAGIC1 1 with primers oriTF-SaF/-CXR. We purified the PCR fragment and inserted it into pGWC 2 by TA cloning, resulting in pGWC-oriTF. We generated ClaI-NotI-GmR-SacII fragment by PCR amplification from pGWG with primers Gen-CNF/Gen-R2. We purified the PCR fragment, digested it with ClaI and SacII, and inserted it into the ClaI and SacII sites of pGWC-oriTF to replace the CmR fragment, resulting in pG-oriTF. We inserted the pheS Gly294 fragment between the SalI and ClaI sites of pML378 1 into the XhoI and ClaI sites of pG-oriTF, resulting in pG-oriTF-pheS. We disrupted the PstI and AatII sites of pheS Gly294 by site-directed mutation with primers pheS-5PF/R phosphorylated at their 5'-end, resulting in pG-oriTF-pheSm. We generated NcoI and XbaI-flanked ApR frament by PCR amplifcation from pUC18 with primers Ap-NcF/-XbR. We purifed the PCR fragment, digested it with NcoI and XbaI, and inserted it into the BspHI and NheI sites of pLC-ccdB 3 , resulting in pLC-Amp. We replaced the sacB fragment of pLC-Amp with the XhoI site by PCR amplification with primers R6K-NXF/sacB-3F. We purified the PCR fragment, digested it with NotI, and allowed it to self-ligate, resulting in pLM2-DsB. We replaced the CmR fragment of pLM2-DsB with the SalI site by PCR amplification with primers MISSA-F/lox22-SaR. We purified the PCR fragment, digested it with SalI, and allowed it to self-ligate, resulting in pLM2-DsB2.
We inserted the SalI-NotI fragment of oriTF-pheSm from pG-oriTF-pheSm into the XhoI and NotI sites of pLM2-DsB2, resulting in pLM2BB. We obtained CmR gene by PCR amplification from pLC-ccdB 3 with primers CmF3/R3, we purified the PCR fragment, and ligated with SalI-digested and T4 pol blunted pLM2BB, resulting in pLC2-A. We inserted the ApaI-PstI fragment of pLACB 3 into the ApaI and PstI sites of pLC2-A, resulting in pLC2-ccdB. We obtained GmR gene by PCR amplification from pGWG 2 with primers Gen-F2/R2, we purified the PCR fragment, and ligated with SalI-digested and T4 pol blunted pLM2BB, resulting in pLG2-A. We disrupted the EcoRV and SacII sites of pLG2-A by inserting a short insert generated by annealing two oligos omEmSF/R into the two sites, resulting in pLG2-A2. We inserted the ApaI-PstI fragment of pRACB 3 into the ApaI and PstI sites of pLG2-A2, resulting in pRG2-ccdB.

Creation of the donor vectors pSL-ccdB and pSR-ccdB
We generated two PCR fragments from pLM2BB: one was flanked by NcoI and XhoI introduced by primers Lox-NcF/RVM21-Xh, the other was flanked by BspHI and XhoI introduced by primers oriTRK2-BsF/oriTF-XhR. We purifed the two PCR fragments, digested them with NcoI/XhoI and BspHI/XhoI, respectively. We ligated the two digested fragments, resulting in pLM2BB-DOP. We obtained NcoI-flanked Em7p:GmR with 94-bp terminator from ApR gene by gene synthesis, we inserted the NcoI fragment into the two BspHI sites of pUC18 to replace ApR gene, resulting in pUC-Em7G. We replaced the GmR ORF of pUC-Em7G with SpR ORF, resulting in pUC-Em7S. We did this by overlaping PCR with two PCR fragments obtained by PCR amplifcation from pUC-Em7G and pMDC150 4 , with primers S-Ater-F/Em7pS-R and Em7pS-F/S-Ater-R, respectively. We introduced XbaI and BspHI sites at the downstream of SpR ORF in pUC-Em7S by PCR amplifcation with primers Ater-BsF/SpR-NhR, we amplified the pheS Gly294 ORF with primers pheS-XbF/-NcR from pRG2-ccdB. We purifed the two PCR fragments, and digested them with BspHI/NheI and NcoI/XbaI, respectively. We purified the digested fragments and allowed them to ligate with each other, resulting in pUC-

Em7SP.
We generated SalI and I-SceI-flanked Em7p-SpR-pheS fragment by PCR amplification from pUC-Em7SP with three primers AiScF/R/iSc-SaFR. We purified the PCR fragment, digested it with SalI and inserted it into the XhoI site of pLM2BB-DOP, resulting in pSLM2BB. We inserted the ApaI-PstI fragment of pLACB 3 into the ApaI and PstI sites of pSLM2BB, resulting in pSL-ccdB. We inserted the ApaI-PstI fragment of pRACB 3 into the ApaI and PstI sites of pSLM2BB, resulting in pSR-ccdB.

Creation of the donor vectors pVLC-ccdB and pVRG-ccdB
We generated XhoI and AflII-SalI-flanked oriV by PCR amplification from pCC1BAC (Epicentre Biotechnologies) with primers oriV-XhF/-ASR, we purified the PCR fragment, digested it with XhoI and AflII, and inserted it into the SalI and AflII sites of pG-oriTF-pheSm, resulting in pG-oriV-TF-pheS. We generated the XhoI and NotIflanked fragment by PCR amplification from pLC2-A with primers oriT-XhF/SacB-3F, we purified the fragment, and digested it with XhoI and NotI. We allowed the digested PCR fragment to ligate with the oriV-oriTF-pheS fragment cut out from pG-oriV-TF-pheS with SalI and NotI, resulting in pVLC-A. We generated pVLG-A in the same manner except that we obtained the PCR fragment from pLG2-A. We inserted the ApaI-PstI fragment of pLACB 3 into the ApaI and PstI sites of pVLC-A, resulting in pVLC-ccdB. We inserted the ApaI-PstI fragment of pRACB 3 into the ApaI and PstI sites of pVLG-A, resulting in pVRG-ccdB.

Creation of the donor vectors for pABA61
To generate pABA61, we constructed donors including pLC2-RCAR11, pRG2-CBF3/NCED3, and pSR-TM1. We obtained the Arabidopsis guard cell promoter GC1 6 by PCR amplification from Arabidopsis genomic DNA with primer pair GC1p-SpF/XbR. We purified the PCR products, digested them with SphI and XbaI, and ligated them with SphI and XbaI-digested pLC2-GUS, resulting in pLC2-GC1p. We obtained the Arabidopsis ABA receptor gene RCAR11 7,8 by PCR amplification from Arabidopsis genomic DNA with primer pair RCAR11-XbF/SaR. We purified the PCR products, digested them with XbaI and SacI, and ligated them with XbaI and SacIdigested pLC2-GC1p, resulting in pLC2-RCAR11. We inserted the HindIII-EcoRI fragments of CBF3/DREB1A 9,10 and NCED3 11 cassettes from pL-CBF3 and pR-NCED3 into the HindIII-EcoRI sites of pRG2-ccdB, respectively, resulting in pRG2-CBF3/NCED3. We inserted the HindIII-EcoRI fragment from pL-TM1 3 into the HindIII-EcoRI sites of pSR-ccdB, resulting in pSR-TM1.

Creation of the donor vectors for pTEST51
To generate pTEST51, we constructed donors including pVLC-Gly and pVRG-gNHX8. We replaced the XbaI-SacI fragment of CmR-ccdB cassette in pMDC32 5 with an insert produced by annealing two oligos oXb-SaF/R, resulting in pMDC32del. We inserted the HindIII-EcoRI fragment of 2x35Sp cassette into the HindIII and EcoRI sites of pVLC-ccdB, resulting in pVLC-2x35S. We obtained CTP2-CP4EPSPS fusion gene harboring an intron from maize HSP70 by PCR amplification from glyphosate-resistance transgenic maize (Monsanto) genomic DNA with primer pair Gly-KpF/-SaR. We purified the PCR products, digested them with KpnI and SacI, and ligated them with KpnI and SacI-digested pVLC-2x35S, resulting in pVLC-Gly-P1.
We replaced the SacI-AscI fragment of the nos terminator in pVLC-Gly-P1 with ocs terminator obtained by PCR amplification from pR-tOcs 3 with primer pair Ocst-SaF/-AsR, resulting in pVLC-Gly. We replaced the XbaI-SacI fragment of GUS gene in pVRG-GUS with an insert produced by annealing two oligos oSm-SaF/R, resulting in pVRG-35S. We inserted the 4.3-kb XbaI-KpnI fragment of genomic NHX8 sequence from pBI121-gNHX8 12 into the XbaI and KpnI sites of pVRG-35S, resulting in pVRG-gNHX8.

Creation of the donor vectors for pGUS-IMF
To generate pGUS-IMF, we constructed donors including pRG2-zCre and pL2-Hyg-ST. We obtained maize codon-optimized Cre gene (zCre) harboring an intron via gene synthesis conducted by GenScript (Nanjing). We inserted the XbaI-SacI fragment of zCre into the XbaI and SacI sites of pRG2-olexA, resulting in pRG2-zCre. We obtained SpR and oriT by PCR amplification from pPZP200 13 and pL-oriT 3 , with primer pairs Sp-AsF/ST-R and ST-F/oriT-PaR, respectively. We mixed the two PCR fragments and performed fusion PCR, with the mixture as templates, and Sp-AsF/oriT-PaR as primer pair. We purified the PCR products, digested them with AscI and PacI, and ligated them with AscI and PacI-digested pLM2-A, resulting in pL-ST.
We replaced the SalI-PacI fragment of pL-ST with an insert produced by annealing oligos oSHEP-F/R, resulting in pL2-ST. We inserted the HindIII-EcoRI fragment of Hyg cassette from pRG2-Hyg into the HindIII and EcoRI sites of pL2-ST, resulting in pL2-Hyg-ST. See above for the construction of pLC2-XVE/GUS, and pSR1-iSc.

Creation of the recipient binary vectors pCB-RTL/LTR
To generate two recipient binary vectors pCB-RTL/LTR, we obtained KmR and oriT by PCR amplification from TAC-RTL 3 with primer pair Kan-SAPF/KT-R, and from pL-oriT 3 with primers KT-F/oriT-SaR, respectively. We mixed the two PCR fragments and performed fusion PCR with the mixture as templates and Kan-SAPF/oriT-SaR as primer pair. We purified the fused PCR products, and ligated them with AflII and SacII-digested and T4 DNA polymerase-blunted pGWC 2 , resulting in pKT. We inserted the AflII-PstI fragment of LB-RB in pGL 14 into the AflII and PstI sites of pKT, resulting in pKT-LR. We obtained loxP-GmR-attR2 cassette by PCR amplification from pLG2-A with primer pair lox-XPF/Amp-R. We purified the PCR products, digested them with HindIII and PmlI, and ligated them with HindIII and PmlI-digested pKT-LR, resulting in pKTG-LTR. In addition, we ligated the HindIII and PmlI-digested PCR products with HindIII and HpaI-digested pKT-LR, resulting in pKTG-RTL. We digested pKTG-LTR with HpaI and PacI, blunted them with T4 DNA polymerase, and re-ligated them, resulting in pKTG-LTR-del. We digested pKTG-RTL with PacI and PmlI, blunted them with T4 DNA polymerase, and religated them, resulting in pKTG-RTL-del. We inserted the SwaI-SalI large fragment of pKTG-LTR-del or pKTG-RTL-del into the HpaI and XhoI sites of pCC1-BAC (Epicentre Biotechnologies), resulting in pCB-LTR/RTL.

Creation of the Agrobacterium helper plasmid pSAH
To generate pSAH, we obtained the trfA and trfB fragments by PCR amplification from E. coli strain BW20676 15 genomic DNA with primer pairs trfA-HiF/-XbR, and trfB-XbF/-EcR, respectively. We mixed the two PCR fragments, purified the PCR products, digested them with HindIII/XbaI/EcoRI, and ligated them with HindIII and EcoRI-digested pLC2-ccdB, resulting in pLC2-trfAB. We obtained the SpR fragment by PCR amplification from pPZP200 13 with primer pair Sp-EcF/AsR. We purified the PCR products, digested them with EcoRI and AscI, and inserted them into the EcoRI and AscI sites of pLC2-trfAB, resulting in pLC2-trfAB-S. We obtained the fragment harboring pVS1-origin and pUC-origin for the plasmid replication in Agrobacterium and E. coli, respectively, by PCR amplification from pPZP200 13 with primer pair pSAH-AsF/PaR. We purified the PCR products, digested them with AscI and PacI, and ligated them with the AscI and PacI-digested pLC2-trfAB-S, resulting in pSAH-trfAB. We obtained the ApR fragment flanked by EcoRI and I-SceI from pUC18 with three primer mixture Ap-iSceF/R/iSce-EcFR. We purified the PCR products, digested them with EcoRI, and inserted them into the EcoRI site of pSAH-trfAB, resulting in pSAH-trfAB-A. We obtained the virG and virE fragment by PCR amplification from pCH32 16 with primer pair virGE-PaF/AsR. We purified the PCR products, digested them with AscI and PacI, and inserted into the AscI and PacI sites of pRG2-ccdB, resulting in pRG2-virGE. We replaced the AflII-PacI region of pRG2-virGE with an insert produced by annealing two oligos oAiSP-F/R, resulting in pVirGE-iSc1. We replaced the AscI-PstI region of pVirGE-iSc1 with an insert produced by annealing two oligos oAsiSPs-F/R, resulting in pVirGE-iSc2. We inserted the I-SceI fragment of virG-virE in pVirGE-iSc2 into the two I-SceI sites of pSAH-trfAB-A, resulting in pSAH.

Creation of the host strains ABO, 254D, 203L, and P254D for suicide donor vectors
We replaced the EcoRI-SpeI region of pDOC-K 17 with an insert produced by annealing two oligos oANXP-F/R, resulting in pDOC-K-del. We obtained bioA and ybhC fragments by PCR amplification from E. coli strain DH10B genomic DNA with primer pairs bioA-XhF/-EHR and ybhC-EcF/-NcR, respectively. We purified the PCR products, digested the purified bioA and ybhC PCR products with EcoRI/XhoI, and EcoRI/NcoI, respectively. We purified the two digested PCR fragments, and ligated the two fragments with NcoI and XhoI-digested pDOC-K-del, resulting in pDOC-YB. We inserted the HindIII-EcoRI fragment of trfA-trfB from pLC-trfAB into the HindIII and EcoRI sites of pDOC-YB, resulting in pDOC-YB-trfAB. We co-transformed pDOC-YB-trfAB and pACBSCE 17 into the E. coli strain SW106 18 . We conducted Gene Doctoring experiments according to the protocol 17 . We incubated the agar plates at 42°C to kill the background cells, resulting in the creation of engineered E. coli strain ABO, in which 6.4-kb trfA-trfB was integrated into the chromosome. We obtained the fragment harboring partial trfA sequence from pLC2-trfAB with primer pair trfA-HiF/trfA-R. We purified the PCR products and cloned the fragment into the blunt-end cloning vector pCBC, resulting in pCBC-trfA. We introduced the 254D mutation into the sequence by PCR amplification of the whole sequence of pCBC-trfA with primer pair trfA254D-5PF/-5PR phosphorylated at their 5'-end. We purified the PCR products and re-ligated them, resulting in pCBC-trfA254D. In the same manner, we obtained pCBC-trfA203L harboring the 203L mutation introduced with primer pair trfA203L-5PF/R. We replaced the HindIII-NdeI region of pDOC-YB-trfAB with the HindIII-NdeI fragment of pCBC-trfA254D/203L, respectively, resulting in pDOC-YB-trfA254D/230L. In the same manner as we generated E. coli strain ABO, we generated the other two strains 254D and 203L.
To generate P254D, we obtained pir mutant gene by PCR amplification from E. coli strain BW23474 15 genomic DNA with primer pair uidA-iSceF/R. We purified the PCR products and cloned the fragment into the blunt-end cloning vector pCBC, resulting in pCBC-pir106L. We found that the pir116 named previously was actually pir106L mutation by sequencing analysis; therefore we re-named the pir copy-up mutant gene pir106L. We obtained ApR fragment flanked by I-SceI from pSAH-trfAB-A with a single primer iSce-FR. We purified the PCR products, and ligated them with NaeI-digested pACBSR 17 , resulting pACBSR-A. We inserted the I-SceI fragment of pCBC-pir106L into the two I-SceI sites of pACBSR-A, resulting in pACBSR-pir106L, which we used thereafter for Gene Doctoring experiments instead of two plasmids pDOC and pACBSCE. We co-transformed the pACBSR-pir106L and pRG2-A2 into the E. coli strain 254D. We conducted Gene Doctoring experiments according to the protocol 17 modified based on one-plasmid strategy. We selected the engineered strain P254D harboring pRG2-A2 on agar plates supplemented with ampicillin and gentamycin. We purged the plasmid pRG2-A2 from the strain by selecting single colonies on Cl-Phe agar plates (0.5% w/v yeast extract, 1% w/v NaCl, 0.4% w/v glycerol, 2% w/v agar, 10 mM D,L-p-Cl-Phe), resulting in plasmid-free E. coli strain P254D.

Creation of the engineered strain DH10B-RV and recipient strains
We cut out Frt-KmR-Frt fragment from pDOC-H 17 with SmaI and SciI, and we ligated the fragment with the MluI-digested and T4 Pol-blunted pCBC-pir106L, resulting in pCBC-FKF, in which KmR was flanked by I-SceI-5'uidA-Frt and Frt-3'uidA-I-SceI. We cut out the I-SceI fragment of KmR from pCBC-FKF with I-SceI, and ligated the fragment with I-SceI-digested pACBSR-A to replace ApR, resulting in pACBSR-FKF. We transformed the pACBSR-FKF into the E. coli strain DH10B and conducted Gene Doctoring experiments according to the protocol 17 , resulting in DH10B-FKF. In this experiment, we used one-plasmid (pACBSR-FKF) strategy to replace twoplasmid strategy (pDOC and pACBSCE). We transformed pCP20 into DH10B-FKF, and allowed Flp/Frt-mediated SSR reaction to occur according to the protocol, resulting in DH10B-Frt. We generated the loxP-GmR-attR2 and CmR-pUC_ori fragments by PCR amplification from pRG2-ccdB and pGWC, with primers LoxGR2-XhF/ccdB-5R and Cm-PaF/pUC_ori-XhR, respectively. We purified the PCR fragments, digested them with XhoI and PacI, and allowed them to ligate with each other, resulting in pGSR-P. We generated SpR fragment by PCR amplification from pPZP200 with primers Sp-F/R, we purified the PCR fragment, and ligated it with the XhoI-digested and T4 Polblunted pLM2BB-DOP, resulting in pSA. We generated ApaI and AflII-flanked SpR-oriTF-R6K fragment by PCR amplification from pSA with primers Sp-ApR/R6K-AfR, we purified the PCR fragment, digested it with ApaI and AflII, and inserted it into the ApaI and AflII sites of pGSR-P, resulting in pGSR-P2. We inserted the Frt site generated by annealing two oligos oFrt-PXF/-PXR into the PacI and XhoI sites of pGSP-P2, resulting in pGSR. We transformed the pGSR into the DH10βF'DOT, we mixed the donor strain harboring pGSR and the heat-shocked recipient strain DH10B-Frt harboring pCP20, and allowed the conjugational transfer and Flp/Frt-mediated SSR reaction to occur, resulting in DH10B-RV.
To generate conjugationally transferrable plasmid harboring two sets of site-specific recombination proteins, we generated the oriTF element by PCR amplification from pMAGIC1with primers oriTF-SpF/BsR, we purified the PCR fragment, digested it with SphI and BstXI, and inserted it into the SphI and BstXI sites of pAH57-Cre, resulting in pAH57-Cre-TF. We transformed the pAH57-Cre-TF into the DH10βF'DOT. We mixed the DH10βF'DOT harboring pAH57-Cre-TF and strains harboring recipient vectors, allowed the conjugational transfer to occur, resulting in recipient strains, including AKG-pCB-LTR/RTL, and AGS-RV.

Creation of the engineered strain DH10B-SRP and recipient strains
We generated N fragment by PCR amplification from SW106 18 genomic DNA with primers N-PiScF0/F and N-ds-HiR, and we generated xis-int fragment by PCR amplification from pAH57-Cre 3 with primers Xis-up-EcF and Int-AiScR0/R. We purified the two PCR fragments, digested them with PacI/HindIII, and EcoRI/AscI, respectively. We ligated the N fragment with PacI and HindIII-digested pLC-Amp, resulting in pLC-NA. We ligated the xis-int fragment with EcoRI and AscI-digested pLC-NA, resulting in pLC-NAX. We digested pLC-NAX with HindIII and EcoRI, blunted the fragment with the T4 Pol, and allowed the fragment to self-ligate, resulting in pLC-NX. We generated the N-xis-int fragment from pLC-NX by digestion with I-SceI, and ligated with the I-SceI-digested pACBSR-A, resulting in pACBSR-

NX.
We transformed pACBSR-NX into the E. coli strain SW106 18 . We conducted Gene Doctoring experiments according to the protocol 17 with modifications: we used oneplasmid strategy (pACBSR-NX) to replace two-plasmids strategy for Gene Doctoring experiments (pDOC and pACBSCE). We incubated the agar plates at 42°C to kill the background cells, resulting in the creation of engineered E. coli strain DH01B-SRP-attL, in which 5.9-kb fragment between N and xis genes was deleted by lambda REDmediated homologous recombination.
We replaced trfA-trfB fragment between the HindIII and EcoRI sites of pDOC-YB-trfAB with an insert produced by annealing with two oligos oHSE-F/R, resulting in pDOC-YB2. We obtained the Frt-KmR-Frt fragment from pDOC-H by digestion with EcoRI and SpeI, and inserted the fragment into the EcoRI and SpeI sites of pDOC-YB2, resulting in pDOC-YB-K. We generated int fragment by PCR amplification from pAH57-Cre with primers int-PaF/HiR, and we generated rrnB-T1T2 by PCR amplification from pLC-ccdB with primers T1T2-HiF/SpR. We purified the two PCR fragments, digested them with PacI/HindIII, and HindIII/SpeI, respectively. We ligated the two fragments with PacI and SpeI-digested pDOC-YB-K to replace the bioA, resulting in pDOC-YI-K. We co-transformed pDOC-YI-K and pACBSCE into the E. coli strain DH01B-SRP-attL. We conducted Gene Doctoring experiments according to the protocol 17 , resulting DH01B-SRP-FKF, in which Frt-KmR-Frt fragment replaced the attL site. We transformed pCP20 into DH10B-SRP-FKF, and allowed Flp/Frt-mediated SSR reaction to occur according to the protocol, resulting in DH10B-SRP. We transformed pCB-RTL/LTR into the DH10B-SRP, resulting in recipient strains, including KG-pCB-LTR/RTL. resulting in pNGG. We amplified the U6-ApR-sgR@Sa cassette with three primers U6-BAF, U6-BEiCR0 and U6-BEiCR from pCBC-U6A@Sa, digested the purified PCR products with BsmBI, and ligated with EcoRI-digested pNGG-P1, resulting in pNNGRRT-P1. We replaced XbaI-SacI fragment of zCas9 in pNNGRRT-P1 with XbaI-SacI fragment of zSaCas9, which was codon-optimized with maize favored codons, synthesized, and cloned into pUC57 by GenScript (Nanjing), resulting in pNNGRRT.

Generation of CRISPR/Cas9 transgenic Arabidopsis and analysis of mutations
We transformed the p2x3sgR into Agrobacterium strain GV3101. We transformed Arabidopsis Col-0 wild-type plants via the floral dip method 22 . We screened the collected seeds from the T0 plants on MS plates containing 25 mg/L hygromycin, and transplanted the resistant seedlings (T1) to soil. We extracted genomic DNA from T1 or T2 transgenic plants grown in soil. To analyze mutations of TRY, CPC and ABI2, we amplified fragments spanning the two target sites of TRY, CPC or ABI2 by PCR using gene-specific primers TRY-5U-F/-3U-R, CPC-5U-F/-3Uds-R, or ABI2-IDF0/-IDR0, respectively. To analyze mutations of ABI1 and HAB1, we amplified fragments spanning the target sites of ABI1 or HAB1 by PCR using gene-specific primers ABI1-IDF0/-IDR0 or HAB1-IDF/-IDR, respectively. To analyze mutations of TRY, CPC, ABI2, and HAB1, we submitted purified PCR products for direct sequencing with the same primers as those for PCR. We analyzed mutations of ABI1 by NcoI digestion analysis of the PCR fragment.
To analyze possible mutations of potential off-target sites of AT5G02760, AT2G25070, and AT3G17090 of the sgRNA targeting ABI1, we amplified fragments surrounding the off-target sites by PCR using gene-specific primers AT5G02760-F/R, AT2G25070-F/R, or AT3G17090-F/R, respectively. We analyzed off-target mutations of AT5G02760 and AT2G25070 by NcoI digestion analysis of the PCR fragments. To analyze mutations of AT3G17090, we submitted purified PCR products for direct sequencing with the same primers as those for PCR.