We transformed the pHEE2A/B/D1/D2/D3/E/F-TRI, pHEN2C-TRI, pHSE2A-TRI, and pHEE2A-CHLI constructs into Agrobacterium strain GV3101, and transformed pHEN2A/B-TRI into GV3101/pSoup [26 (link)]. We transformed Arabidopsis Col-0 wild-type plants via the floral dip method [45 (link)]. 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 transgenic plants grown in soil. To analyze mutations of TRY, CPC, and ETC2, we amplified fragments surrounding the target sites of TRY, CPC, or ETC2 by PCR using gene-specific primers TRY-IDF0/R0, CPC-IDF0/R0, or ETC2-IDF0/R0, respectively [26 (link)]. We submitted purified PCR products for direct sequencing with primers TRY/CPC/ETC2-seqF [26 (link)] located within the PCR fragments. To analyze possible mutations of potential off-target sites of TRY, CPC, and AT5G50230 of the sgRNA targeting ETC2, we amplified fragments surrounding the off-target sites by PCR using gene-specific primers TRY-off-IDF/R, CPC-off-IDF2/R, or 5G50230-off-IDF/R, respectively. We submitted purified PCR products for direct sequencing (as opposed to sequencing of individual clones of PCR products) with primers TRY/CPC/5G50230-off-seqF located within the PCR fragments. To analyze mutations of CHLI1 and CHLI2, we amplified fragments surrounding the target sites of CHLI1 or CHLI2 by PCR using gene-specific primers CHLI1-IDF/R or CHLI2-IDF/R, respectively. We submitted purified PCR products for direct sequencing with primers CHLI1/2-seqF located within the PCR fragments. We then cloned poorly sequenced PCR products, and submitted individual positive clones for sequencing using the T7 primer. To screen the segregated non-transgenic T2 plants, we first screened nine primer combinations, with three forward primers including zCas9-IDF3-2/-IDF5/-IDF6 (located at zCas9) and three reverse primers including rbcS_E9t-IDR/-IDR2 (located at rbcS-E9 terminator) and lacp-IDF (located at the lac promoter of the vector backbone), for more specific primers (Additional file 2: Table S3). We obtained three more specific primer pairs, including zCas9-IDF3-2/rbcS_E9t-IDR2, zCas9-IDF5/lacp-IDF, and zCas9-IDF6/lacp-IDF, with wild-type genomic DNA serving as a negative control and genomic DNA from T1 transgenic plants serving as a positive control (Additional file 2: Table S3). We then performed counterselection PCR with the three primer pairs for screening of non-transgenic T2 plants.
Free full text: Click here