Fig. 2

Generation of SF3B1 variants using the CDE platform. a Agrobacterium-mediated transformation was conducted using the sgRNA library targeting SF3B1. After selection on hygromycin, regeneration was performed under selection pressure of GEX1A (0.4 or 0.6 μM). A non-specific sgRNA was transformed and used as GEX1A selection control. Regeneration was only observed with the sgRNA library targeting SF3B1. Red arrows indicate the GEX1A-resistant shoots. b The resistant plants genotyped by Sanger sequencing and revealed in-frame mutations in SF3B1. These mutants were named SGR (SF3B1 GEX1 Resistant). The red letters indicate the amino acids modified in mutant sequence. SGR1 has a deletion of Q157. SGR2 has a deletion of ten amino acids DAPDATPGIG (223–232). SGR3 has a deletion of K1050. The chromatograms show Sanger sequencing of SF3B1 mutant variants. c A protein domain-focused CDE platform used to generate SF3B1 mutant variants resistant to GEX1A. SGR4 has three consecutive substitutions K1049R, K1050E, and G1051H. SGR5 has the H1048Q substitution and K1049 deletion. SGR6 has the H1048Q substitution, K1049 deletion, and A1064S substitution. SGR4 and SGR5 were recovered with the sgRNA HR target while SGR6 was recovered with PTG transformation