Isolation of CRISPR-Cas-9-induced mutants of MIR172A-E.

(A–E) The positions of each sgRNA are indicated (blue indicates a functional sgRNA, red indicates a non-functional sgRNA, according to the T7E1 assays). The miR172^* and miR172 coding sequences are highlighted in red and orange, respectively. The PAM sequence required for functionality of the Cas-9-sgRNA complex is highlighted in purple. (F–H, J–L) Agarose gels of the indicated T7E1 assays of functional sgRNAs. DNA was derived from T₁ plants transformed with the CRISPR-Cas-9 system from [53]. The expected patterns of restriction digest are indicated below the photograph, and the associated sgRNA is indicated above. (I) An agarose gel of a PCR of MIR172C using DNA derived from T₁ plants harboring the CRISPR-Cas-9 system from [52], indicating the presence of a deletion (asterisk). Data underlying panels F to L are provided in S3 Data. (M) The sequences of WT MIR172A-E genes and the mutants identified by CRISPR-Cas-9. The miR172^* and miR172 coding sequences are highlighted in red and orange, respectively, while the sequences required for the first processing cleavages by DCL1 are highlighted in green. Note that mir172d-3 contains several SNPs (highlighted in red) rather than a deletion. (N) An example of a PPT-resistance assay to identify plants that lack the Cas-9-containing T-DNA. Young leaves were places on an MS-agar (-sucrose) plate supplemented with 25 μg/mL PPT and incubated in a growth chamber for 3 days. The leaves derived from plants lacking the Cas-9-containing T-DNA are light green (red circles), whereas the leaves derived from plants harboring the Cas-9-containing T-DNA are dark green. Cas-9, CRISPR associated protein-9; CLN, cauline leaf number; CRISPR, clustered regularly interspaced short palindromic repeats; PAM, protospacer adjacent motif; PPT, phosphinothricin; sgRNA, single guide RNA; WT, wild-type.

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