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Fig. 1 | Genome Biology

Fig. 1

From: CRISPR directed evolution of the spliceosome for resistance to splicing inhibitors

Fig. 1

The CRISPR/Cas-directed evolution (CDE) platform. a All possible sgRNAs targeting the whole coding sequence of a gene are designed. b The sgRNA library is constructed via oligo synthesis and annealing. c The annealed oligos are cloned with sgRNA scaffold under the U3 promoter in the binary vector. The sequences are confirmed by Sanger sequencing. d All the plasmids are pooled in equimolar ratios. e The pooled plasmids are transformed into Agrobacterium. f The Agrobacterium cells are washed from plates with transformation medium and used for callus transformation. g After two consecutive selections on hygromycin, the callus is regenerated under selection pressure (e.g., splicing inhibitor). h The resistant seedlings are recovered. i The resistant plants are further analyzed by exhaustive phenotyping under selection pressure. The plants are genotyped by amplicon sequencing, and protein variants are identified

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