CRISPR directed evolution of the spliceosome for resistance to splicing inhibitors

Abstract

Increasing genetic diversity via directed evolution holds great promise to accelerate trait development and crop improvement. We developed a CRISPR/Cas-based directed evolution platform in plants to evolve the rice (Oryza sativa) SF3B1 spliceosomal protein for resistance to splicing inhibitors. SF3B1 mutant variants, termed SF3B1-GEX1A-Resistant (SGR), confer variable levels of resistance to splicing inhibitors. Studies of the structural basis of the splicing inhibitor binding to SGRs corroborate the resistance phenotype. This directed evolution platform can be used to interrogate and evolve the molecular functions of key biomolecules and to engineer crop traits for improved performance and adaptation under climate change conditions.

Keywords: CRISPR/Cas9; Directed evolution; Genome engineering; Herbicide resistance; Herboxidiene; Pladienolide B; SF3B complex; SF3B1; Spliceosome; Spliceostatin A; Splicing modulators.

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

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

Fig. 3

SGRs confer resistance to GEX1A treatment and the structural basis of resistance. a Dose–response effects of GEX1A treatment on germination of WT, SGS, SGR1, SGR2, SGR3, SGR4, SGR5, and SGR6 seeds. Rice seeds were sterilized and germinated in dH₂O with different concentrations of GEX1A. Hypocotyl emergence was considered as germination. We observed that the rice seed germination was affected in a dose-dependent manner (Additional file 1: Figure S1). Germination of wild-type and SGS1 seeds was severely inhibited at 2.5 and 5 μM GEX1A. Germination of SGRs is less affected by GEX1A treatment. SGR4 germination is completely unaffected even at 10 μM GEX1A (n = 8). b The structural basis of the SF3B1 mutant variant resistance to GEX1A. The 3D model for the OsSF3B1:PHF5A complex and GEX1A with OsSF3B1 is represented in gray, PHF5A is represented in cyan, and GEX1A represented in blue. Key residues on PHF5A are shown in orange whereas residues for wild type are shown in magenta and mutations on OsSF3B1 are shown in gray

Fig. 4

GEX1A treatment inhibits pre-mRNA splicing in WT rice but not the SGR4 mutant. The cDNAs were prepared from 1-week-old rice seedlings that were treated with 0.3 μM GEX1A for 6 h. RT-PCR was performed using primers that flank alternatively spliced introns in selected genes. Arrowheads indicate splicing variants that changed following GEX1A treatment. The gene structure flanking the amplified fragments and the structures of regulated variants are shown under the gel images (the green line marks the positions of the PCR product). Blue box, exon; line, intron; white box, 5′ or 3′ UTR. The gene locus identifier is shown on the bottom