Targeted saturation mutagenesis of crop genes could be applied to produce genetic variants with improved agronomic performance. However, tools for directed evolution of plant genes, such as error-prone PCR or DNA shuffling, are limited1. We engineered five saturated targeted endogenous mutagenesis editors (STEMEs) that can generate de novo mutations and facilitate directed evolution of plant genes. In rice protoplasts, STEME-1 edited cytosine and adenine at the same target site with C > T efficiency up to 61.61% and simultaneous C > T and A > G efficiency up to 15.10%. STEME-NG, which incorporates the nickase Cas9-NG protospacer-adjacent motif variant, was used with 20 individual single guide RNAs in rice protoplasts to produce near-saturated mutagenesis (73.21%) for a 56-amino-acid portion of the rice acetyl-coenzyme A carboxylase (OsACC). We also applied STEME-1 and STEME-NG for directed evolution of the OsACC gene in rice and obtained herbicide resistance mutations. This set of two STEMEs will accelerate trait development and should work in any plants amenable to CRISPR-based editing.
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The authors declare that all data supporting the findings of this study are available in the article and its supplementary figures and tables or are available from the corresponding author upon request. For sequence data, rice LOC_Os IDs LOC_Os01g55540 (OsAAT), LOC_Os05g22940 (OsACC), LOC_Os03g05730 (OsCDC48), LOC_Os09g26999 (OsDEP1), LOC_Os02g11010 (OsOD, OsEV) are available on the Rice Genome Annotation Project (http://rice.plantbiology.msu.edu/). The deep sequencing data have been deposited with the NCBI BioProject database under accession code numbers PRJNA590652 and PRJNA590653. Plasmids encoding STEME-1, STEME-2, STEME-3, STEME-4, STEME-NG, A3A-PBE-NG, PABE-7-NG, pH-STEME-1-esgRNA and pH-STEME-NG-esgRNA will be available from Addgene.
The custom Python script to analyze types of mutational reads and amino acid substitutions is in the Supplementary Code file.
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We thank S. Jin for technical support on bioinformatic analysis. This work was supported by grants from the Strategic Priority Research Program of the Chinese Academy of Sciences (Precision Seed Design and Breeding, grant no. XDA24000000), the National Natural Science Foundation of China (grant nos. 31788103 and 31900301) and the National Key Research and Development Program of China (grant no. 2016YFD0101804).
The authors have submitted a patent application based on the results reported in this paper.
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Integrated supplementary information
(a)-(f) are OsAAT, OsACC, OsCDC48, OsDEP1, OsEV, and OsOD targets, respectively. One of three independent experiments is shown.
(a) The allelic outcomes of rice sites edited by STEME-1 (n=1 shown) in rice protoplasts. The simultaneous C>T and A>G editing efficiency of STEME-1 was ranged from 0.68-15.37% vs. 0.69-15.50% while only count the A>G substitutions (including single A>G substitution and simultaneous C>T and A>G substitutions) (Fig. 1d–f). (b) Indel frequencies of STEMEs and Cas9 in rice protoplasts. Comparison of indel frequencies by the A3A-PBE, PABE-7, STEME-1, STEME-2, STEME-3, and STEME-4 constructs in rice protoplasts. Values and error bars indicate the mean ± s.e.m of three independent experiments.
(a) Architectures of the A3A-PBE-NG, PABE7-NG and pCas9-NG. ecTadA7.10, evolved Escherichia coli TadA; aa, amino acid; XTEN, a 16-aa linker; NLS, nuclear localization signal; CaMV, cauliflower mosaic virus; Term, terminator. (b) Sequences of 16 NG PAM targets in four loci of the rice genome. The PAM sequences are shown in red. (c) Diagram of OsAAT, OsCDC48, OsDEP1 and OsODEV, and their NG PAM targets. Regions flanking about 80bp of the OsAAT, OsCDC48, OsDEP1 and OsODEV loci were selected to design 20-nt sgRNA sequences to reduce the possible influence of chromatin state on editing.
(a)-(p) are NG PAM targets in OsAAT, OsCDC48, OsDEP1, and OsODEV, respectively. One of three independent experiments is shown.
Both A3A-PBE-NG (a) and PABE7-NG (b) had broad capacities for editing the targets of NGA, NGT, NGC or NGG PAMs. An untreated protoplast sample served as control. Values and error bars indicate the mean ± s.e.m of three independent experiments.
Supplementary Fig. 6 Indel frequencies of A3A-PBE-NG, PABE7-NG and STEME-NG compared with Cas9-NG in rice protoplasts.
A3A-PBE, PABE-7, STEME-1 and Cas9 together with NGG PAM sgRNAs served as positive control. The resulting efficiencies were log scaled by the base number 10. An untreated protoplast sample served as a negative control. Values and error bars indicate the mean ± s.e.m of three independent experiments.
Supplementary Fig. 7 Saturated mutagenesis of a 168-bp region of OsACC by A3A-PBE-NG in rice protoplasts.
A3A-PBE-NG (a) and the untreated control (b) are shown. Max values were calculated when different targets converted the same cytosine and guanine. Values and error bars indicate the mean ± s.e.m of three independent experiments.
Supplementary Fig. 8 C>T and A>G simultaneous editing and indel frequencies of 20 OsACC targets in rice protoplasts.
(a) The simultaneous editing frequencies of C>T and A>G in 20 targets of OsACC by STEME-NG in rice protoplasts. (b) Indel frequencies of 20 OsACC targets by A3A-PBE-NG, STEME-NG and Cas9-NG in rice protoplasts. An untreated protoplast sample served as a negative control. Values and error bars indicate the mean ± s.e.m of three independent experiments.
Supplementary Fig. 9 Saturated substitutions of 56 amino acids in the OsACC CT domain by A3A-PBE-NG in rice protoplasts.
Frequencies of silent, missense, and nonsense mutations were collected.
Supplementary Fig. 10 An overview of sgRNAs for saturated substituting 400 amino acids in the OsACC CT domain.
(a) An overview of the saturated targets on OsACC CT domain for rice plants. (b) Architectures of the binary vectors pH-STEME-1-esgRNA and pH-STEME-NG-esgRNA. ecTadA7.10, evolved Escherichia coli TadA; aa, amino acid; XTEN, a 16-aa linker; NLS, nuclear localization signal; E9 Term, pea rbcS-E9 terminator; pd35S, cauliflower mosaic virus double 35S promoter; Hyg, hygromycin. Arrows indicate the primers for amplicon deep sequencing.
(a) Base editing efficiencies of STEMEs in each transformed group. (b) Indel efficiencies of STEMEs in each transformed group. An average 0.90% indel reads in these groups, and 31.47% of the indels conferred in-frame mutations (Supplementary Dataset 2). Wild-type plants were used as untreated controls.
(a) The P1927F and Q1926* changes were caused by C:G>T:A transitions. Asterisk represents the stop codon. (b) The A2123T and W2125C changes were caused by a C:G>T:A transition and a C:G>G:C transversion, respectively. (c) The S1866F change was caused by a C:G>T:A transition and a silent mutation of G1869 was the result of a C:G>T:A transition. (d) The A1884P change was caused by a C:G>G:C transversion (A1884P-Hetero1) or simutaniously C:G>G:C transversion and A:T>G:C transition (A1884P-Hetero2). The edited bases are shown in lower case. Red triangles indicate peaks of edited bases. The genotypes of these mutations with two double peaks were confirmed further by T-vector cloning.
(a) Schematic of the procedure for mutating the OsACC CT domain via STEMEs on medium supplemented with haloxyfop using Groups 6 and 20 of individual sgRNAs. (b-c) The percentage of sgRNA sequencing reads after selection on medium containing 0.108 mg l-1 haloxyfop for 4 weeks in Group 6 (b) and Group 20 (c). The sgRNA OsACC-sg33 covered P1927 in Group 6 accounts for 100%, OsACC-sg140 and OsACC-sg141 both covered W2125 in Group 20 account for 96.74%. For the mutational reads of target region, 63.53% mutated sequences involved P1927F in Group 6, 48.71% mutated sequences involved W2125C in Group 20 (Supplementary Dataset 3). (d) Herbicide resistance seedlings harboring P1927F and W2125C mutations, respectively, regenerated in haloxyfop-containing medium. One biological experiment was performed. Scale bar, 2 cm. (e) Genotypes of herbicide resistance seedlings from Group 6 and Group 20. The P1927F and Q1926* were caused by C:G>T:A transitions; the A2123T was caused by C:G>T:A transition or C:G>T:A and A:T>G:C simultaneously conversions; the W2125C changes were caused by a C:G>G:C transversion. The edited bases are shown in lower case. Asterisk represents the stop codon. Red triangles indicate peaks of edited bases. The genotypes of these mutations with two double peaks were confirmed further by T-vector cloning.
Supplementary Fig. 14 Sequencing of OsACC full-length genomic DNA of P1927F, W2125C, S1866F, and A1884P mutants.
Twenty-two paired primers were used to sequence OsACC genomic DNA. The base changes of each mutant in P1927F, W2125C, S1866F, and A1884P codons were indicated. No other mutational changes occurred except for these target sites.
(a) Phenotypes of two homozygous A2123T mutants. One biological experiment was performed. Scale bar, 2 cm. Red arrows indicate the died symptom of newly grown leaves. (b) Genotypes of A2123T mutants. The A2123T change was caused by a C:G>T:A transition. The edited bases are shown in lower case. Red triangles indicate peaks of edited bases.
Supplementary Figs. 1–15, Supplementary Tables 1–8, Supplementary Sequences 1–3 and Supplementary Code.
Details of the saturated mutagenesis of 56 amino acids of the OsACC CT domain in rice protoplasts.
Mutational reads of directed evolution of 400 amino acids of the OsACC CT domain in rice plants.
Mutational reads of using STEMEs for targeted mutagenesis under concurrent selection pressure.
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Li, C., Zhang, R., Meng, X. et al. Targeted, random mutagenesis of plant genes with dual cytosine and adenine base editors. Nat Biotechnol (2020). https://doi.org/10.1038/s41587-019-0393-7