Choosing T1 progeny after CRISPR editing in rice
Rice transformation produced 150 independent T0 lines carrying Cas9, an sgRNA targeting OsGENE, and a hygromycin resistance cassette. Deep amplicon sequencing of T0 leaf DNA gives: line A, 62% reads with a 1-bp insertion and 5% wild type; line B, 96% reads with a 4-bp deletion; line C, 34% edited reads with three different indels; line D, no editing. qPCR shows line A has one T-DNA copy, line B one, line C three. Only 22 of 105 edited T0 lines show the expected dwarf phenotype. Line B is the strongest phenotype but carries a 4-bp deletion predicted to cause a frameshift at codon 180 of 620. The breeder wants to advance line B immediately to a yield trial and use line A as a backup. The greenhouse has space for 96 T1 plants per line. No off-target edits were detected in the top 10 predicted sites for either line.
1. Interpret the editing data. Can line B be advanced directly to a yield trial? What T1 genotyping is required?
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Line B is the best candidate: 96% of reads carry one defined 4-bp deletion, it has a single T-DNA copy, the deletion is predicted to frameshift at codon 180, the phenotype is strong, and no off-target edits were found among the top 10 sites. However, a T0 plant cannot go directly to a yield trial as a finished genotype. T0 plants are regenerated from callus and may be chimeric; the 4% wild-type reads could come from a wild-type allele, from non-edited sectors, or from sequencing noise. The T-DNA and Cas9 are also still segregating, and the dwarf phenotype could be affected by somaclonal variation or tissue culture effects.
T1 genotyping must confirm: the exact 4-bp deletion by amplicon sequencing or a cleavage assay; homozygosity versus heterozygosity; absence of the Cas9 T-DNA by PCR; single-copy T-DNA by qPCR; no new off-target edits at the validated sites; and co-segregation of the deletion with the dwarf phenotype. Line A is chimeric or mosaic (62% edited, 5% wild type) and is not a clean backup until its T1 progeny are analysed. Line C, with 34% editing and three T-DNA copies, should be discarded.
2. If line B is biallelic but heterozygous (one mutant and one wild-type allele), calculate the T1 plants needed to find at least 10 homozygous mutants with 95% probability. State assumptions.
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For a single Mendelian locus, selfing a heterozygote gives 1/4 homozygous mutant, 1/2 heterozygous and 1/4 wild type. Thus p = 0.25 for a homozygous mutant in each T1 plant. We need the smallest n such that P(X at least 10) is at least 0.95, where X is binomial(n, 0.25). Using the normal approximation with continuity correction: 0.25n - 1.645 sqrt(0.1875n) = 10. Solving gives n of about 62-63; a safe answer is 63 T1 plants. Exact binomial calculation puts the 95% point at about 62, so 63 provides a small margin. The 96-plant greenhouse space is therefore sufficient.
Assumptions: one independently segregating locus; no segregation distortion or gamete lethality; the T0 is heterozygous with one mutant and one wild-type allele; all T1 plants survive to genotyping; and the phenotype does not require homozygosity for viability. If line B is actually homozygous for the deletion, all T1 plants should carry the mutant allele and only about 10 plants are needed for confirmation.
3. Why did only 22 of 105 edited T0 lines show the expected dwarf phenotype? Give two explanations and a check.
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Explanation 1: many edits are in-frame or do not disrupt the functional domain. A 1-bp or 3-bp indel can preserve the reading frame, and edits near the ends of the protein may retain partial function. The phenotype may also require biallelic or homozygous disruption, which many T0 lines do not yet have. Check: classify each line by allele type (frameshift versus in-frame), edit position, and zygosity, then compare phenotype frequency among classes.
Explanation 2: the phenotype is masked or modified by chimerism, T-DNA position effects, somaclonal variation, or greenhouse conditions. A line with only 34% edited reads may have large non-edited sectors, and a line with three T-DNA copies may show silencing or stress. Check: test T1 progeny for co-segregation of the confirmed homozygous deletion with dwarfism, measure OsGENE transcript and protein in young leaves, and compare independent T1 families grown side by side under the same conditions. If the phenotype does not co-segregate with the deletion in T1, it is not a clean gene-edited phenotype.