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. 2018 May 24;9:699. doi: 10.3389/fpls.2018.00699

FIGURE 3.

FIGURE 3

The predicted off-targets of three USTA gRNA spacers. (A) The sequences of three USTA gRNA spacers and numbers of their off-targets with 1–5 nucleotide differences. The off-target sites of gRNA spacers UA01, UA13, and UA21 were predicted with the Cas9off program. All of them have no off-targets with only one or two nucleotide variations. Spacer UA01 has only one off-target with four nucleotide variations. (B) Sequences of the off-targets of with fewer than five nucleotide variations from USTA gRNA spacers and experimental verification results. Nucleotides in the off-targets differing from the gRNA sequences are in pink. The PAM sequences (NGG) are in blue. For each gRNA spacer, the off-target sites were amplified and sequenced from 10 corresponding ustA deletion mutants. No mutations were detected in any of the 10 ustA mutants (0/10) at UA13-3-1, UA13-4-2, UA13-4-3, UA21-3-1, and UA21-4-1. NA, no data available due to PCR failures.