Gene editing by co-transformation of TALEN and chimeric RNA/DNA oligonucleotides on the rice OsEPSPS gene and the inheritance of mutations

Abstract

Although several site-specific nucleases (SSNs), such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas, have emerged as powerful tools for targeted gene editing in many organisms, to date, gene targeting (GT) in plants remains a formidable challenge. In the present study, we attempted to substitute a single base in situ on the rice OsEPSPS gene by co-transformation of TALEN with chimeric RNA/DNA oligonucleotides (COs), including different strand composition such as RNA/DNA (C1) or DNA/RNA (C2) but contained the same target base to be substituted. In contrast to zero GT event obtained by the co-transformation of TALEN with homologous recombination plasmid (HRP), we obtained one mutant showing target base substitution although accompanied by undesired deletion of 12 bases downstream the target site from the co-transformation of TALEN and C1. In addition to this typical event, we also obtained 16 mutants with different length of base deletions around the target site among 105 calli lines derived from transformation of TALEN alone (4/19) as well as co-transformation of TELAN with either HRP (5/30) or C1 (2/25) or C2 (5/31). Further analysis demonstrated that the homozygous gene-edited mutants without foreign gene insertion could be obtained in one generation. The induced mutations in transgenic generation were also capable to pass to the next generation stably. However, the genotypes of mutants did not segregate normally in T1 population, probably due to lethal mutations. Phenotypic assessments in T1 generation showed that the heterozygous plants with either one or three bases deletion on target sequence, called d1 and d3, were more sensitive to glyphosate and the heterozygous d1 plants had significantly lower seed-setting rate than wild-type.

Fig 1. Chimeric RNA/DNA oligonucleotides (COs).

Uppercase letters represent DNA residues, lowercase letters represent 2′-O-methyl-RNA residues, and the box indicates the nucleotide that should be introduced into the target sequence.

Fig 2. TALEN discrimination sequence and vector construction.

(A) pCAMBIA1301M for plant transformation. (B) TALEN left-arm cassette L20. (C) TALEN right-arm cassette R16. (D) The discrimination sequence and target site in rice genome. The asterisk indicates the target base C³¹⁷ and the box indicates the spacer sequence.

Fig 3. Sequencing maps of the PCR product of some calli lines in the T0 generation.

4–1 is a chimera, 4–3 is a homozygote, and 4–14, 4–19, and 6–5 are heterozygotes. The sequence between two arrows is the spacer cut by TALEN. The scissors indicate three-base deletions and the asterisk indicates the targeted base substitution C³¹⁷-T.

Fig 4. Mutations from the calli flow to the regenerated plants.

The genotype and detailed sequence variation of some typical mutants from chimera, homozygote and heterozygote are presented. Each dashed line represents a deleted nucleotide. The nucleotide in gray represents substitution. WT, wild-type sequence; d, base deletion; s, base substitution. The number after ‘d’ or ‘s’ represents the number of bases that have been deleted or substituted. The box marks the variant sequence which might result from mismatch repair in E. coli during PCR product cloning.

Fig 5. The segregation patterns of TALEN construct and the OsEPSPS genotype from T0 to T1 generation in some transgenic lines.

T1 population are divided into two groups according to whether the TALEN construct exists (+) or not (–) in the genome. The number before genotype indicates the amount of T1 plants and the number after ‘d’ indicates the amount of bases that have been deleted. WT, wild-type sequence; d, base deletion.

Fig 6. Phenotypic assessments of heterozygous OsEPSPS mutants with either one base deletion (WTd₁) or three bases deletion (WTd₃) and wild-type plant (WTWT) in T1 generation.

A. Glyphosate test (Roundup 300×dilution). Heterozygous mutants are more sensitive to glyphosate; B. Spikelets; C. Seed-setting rate. Bar = 10 cm.