Fig. 5: Off-target effects of CRISPR TiD in tomato.
From: Genome editing in plants using CRISPR type I-D nuclease
a, b TiD target site numbers including mismatches in the SlIAA9 and SlRIN genes (a) and the tomato, Arabidopsis, and rice chromosomes (b). The target sites for SpCas9 (PAM; NGG) and MaTiD (PAM; GTH) were counted using Cas-OFFinder and an in-house Perl script. c Upper; The off-target sequences of SlIAA9 GTC_gRNA1(+) target sequence. Red characters; miss-matched nucleotides. Green boxes; PAM. Lower; mutation efficiencies in the cloned PCR products of off-target sites from the transgenic T0 shoots of Ailsa Craig (SlIAA9-tid_gRNA1(+) AC T0s_ #1, 2, and 4) and wild-type (WT) were calculated from the read counts in the deep amplicon sequencing by Mi-seq. d Upper; Off-target sequences of SlRIN GTC_4003–4238(+) target sequence. Red characters; mis-matched nucleotides. Green boxes; PAM. Lower; Long-range PCR (3 kbp + 3kbp) at off-target sites in transgenic T0 shoots of Micro-Tom (SlRIN-tid_ GTC_4003–4238(+)MT T0s_ #1–12), wild-type (WT) and vector control plants (VC).
