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Table 3 Mutation analysis of likely triple mutants segregated from two representative T1 lines with normal phenotypes

From: Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation

Line ETC2 mutation TRY mutation CPC mutation Genotype
T1 #4 0/0 +G (×3)//+A (×9)//−G (×7)//+C (×2) 0/0 EEtxCC
T2 4-1 +C/+T −G/−G +A/+A eettcc
4-2 +A/+A −G/−G −8 (×8)/−G + T (×2) eettcc
4-3 +T (×9)/+5 (×11) +A (×7)/−5 (×3) +G (×4)/−14 + 4 (×6) eettcc
4-4 −41 (×9)/−24 (×11) −G/−G −G/−G eettcc
4-5 −14 + A (×13)/+A (×7) −G/−G −3/−3 eettcc
4-6 −5/−5 +G (×8)/−G (×2) +G/+A eettcc
4-7 +T/+A +C/+C +G/+T eettcc
4-8 +T/+T −G/−G +G/+A eettcc
4-9 +C/+T −G (×7)/−8 (×3) +G/+G eettcc
4-10 +G/+G +T (×3)/−G (×7) 0 (×13)//+G (×2)//+A (×1)//+T (×2) eettcx
T1 #6 +A (×2)//−5 (×1)//−41 (×2)//−52 + 9 (×3)//−24 (×2) 0 (×4)//+G (×5)//−G (×8)//−22 + 16 (×5) 0/0 extxCC
T2 6-1 −52 + 9/−52 + 9 −22 + 16(×6)/−3(×5) 0 (×7)//+T (×1)//+A (×1) eettcx
6-2 +T/+T −G (×10)//+G (×5)//−26 (×4) 0 (×15)//+A (×2)//+T (×1)//−14 + 4 (×1)//−11 (×2) eetxcx or eettcx
6-3 −52 + 9/−52 + 9 +T (×5)//+G (×3)//−G (×2)//−22 + 16 (×3) +A (×5)/+A (×5) eetxcc or eettcc
6-4 −52 + 9/−52 + 9 −22 + 16/−22 + 16 +G/+A eettcc
6-5 −52 + 9/−52 + 9 −22 + 16 (×5)/+G (×3) +A/+T eettcc
6-6 −41 (×5)//−24 (×3)//−30 + 17 (×2)//+A (×2) −G (×8)//+G (×2)//−22 + 16 (×1) +A (×1)/+T (×8) extxcc or eettcc
6-7 −4 (×16)/+G (×4) −G/−G −G (×9)/+C (×2) eettcc
6-8 +C (×10)/−49 (×10) −G/−G +T/+T eettcc
6-9 −52 + 9/−52 + 9 +A (×5)//−G (×2)//−22 + 16 (×1) +A/+T eetxcc or eettcc
6-10 +A/+T −G/−G +T/+T eettcc
  1. ‘+’ indicates insertion, ‘–’ indicates deletion, ‘0’ indicates no mutation (wild-type allele). The number following ‘+’ or ‘–’ indicates the number of bases inserted or deleted; if the number is 1, it is replaced with a specific base. Mutations were detected by direct sequencing of PCR products or sequencing of cloned PCR products. Two types of mutations from direct sequencing of PCR products were obtained based on double-peaks on a chromatograph. When mutations were detected by sequencing of cloned PCR products, the number of the same type of mutation is indicated in parentheses. Two alleles (in WT, homozygous or biallelic mutants, or heterozygous mutants) are separated by ‘/’, whereas more than two alleles (in mosaic plants, underlined) are separated by ‘//’ between two alleles. For genotypes, E/T/C corresponds to the wild-type ETC2/TRY/CPC gene, e/t/c corresponds to etc2/try/cpc mutant gene, x corresponds to multiple alleles, resulting in mosaic plants