Yanmin Lia, Jinjie Zhua, Hao Wua, Changlin Liua, Changling Huanga, Jinhao Lanb, Yanming Zhaob, Chuanxiao Xie

ScienceDirect; 2019 Crop Science Society of China and Institute of Crop Science (Accepted 10 November 2019 Available online)

ABSTRACT

Single-nucleotide polymorphisms contribute to phenotypic diversity in maize. Creation and functional annotation of point mutations has been limited by the low efficiency of conventional methods based on random mutation. An efficient tool for generating targeted single-base mutations is desirable for both functional genomics and precise genetic improvement. The objective of this study was to test the efficiency of targeted C-to-T base editing of two non-allelic acetolactate synthase (ALS) in generating sulfonylurea herbicide-resistant mutants. A CRISPR/Cas9 nickase-cytidine deaminase fused with uracil DNA glycosylase inhibitor (UGI) was employed to achieve targeted conversion of cytosine to thymine in ZmALS1 and ZmALS2. Both protoplasts and recovered mutant plants showed the activity of the cytosine base editor, with an in vivo efficiency of up to 13.8%. Transgene-free edited plants harboring a homozygous ZmALS1 mutation or a ZmALS1 and ZmALS2 double mutation were tested for their resistance at a dose of up to 15-fold the recommended limit of chlorsulfuron, a sulfonylurea herbicide widely used in agriculture. Targeted base editing of C-to-T per se and a phenotype verified in the generated mutants demonstrates the power of base editing in precise maize breeding.

See: file:///D:/PDF%20files/Maize/1-s2.0-S2214514119301461-main.pdf

Figure 2: Targeted base editing of ZmALS1 conferred chlorsulfuron herbicide resistance in maize. (A) The target site for ZmALS by CT-nCas9. The target sequence of ZmALS-sgRNA is shown and the PAM DNA sequences are underlined. Gray-shaded sequences indicate an introduced NruI restriction site derived from the expected edited C7 → T7 substitution mutation. The nucleotide number is counted from the end distal to the protospacer-adjacent motif (PAM) as position 1 in the sgRNA associated region). T(G), T SNP at −17 site indicates ALS1 and G SNP indicates ALS2 allele. (B) PCR-RE (NruI) assay for efficient identification of T2 homozygous C7 → T7 substitution mutant plants. Only the homozygous mutation sequences were cleaved into two bands; the heterozygous mutations sequence were partially cleaved. Other mutations and the wild type were not cleaved. +, positive control of artificial PCR created C7 → T7 single based substitution mutation M, DNA size marker; WT, wild type negative control. (C) The observed four different base-edited mutations and their frequencies among the 16 mutant plants. The expected target C → T base editing activity window is indicated in light blue shading. (D) Chlorsulfuron resistance experiment among T2 mutant individuals (on left in pods) harboring homozygous C7 → T7 mutation and wild type plants (on right in pods) in six-leaf-stage seedlings. Scale bar, 10 cm. (E) In vitro specific ALS activity was characterized in mutant and wild-type plants using 10 nmol L−1 chlorsulfuron.