Fig. 4: CRISPR-GuideMap streamlines phenotype discovery in CRISPR Libraries.
a Schematic visualization of the CRISPR-GuideMap system. DNA from each plant is amplified using primers with unique 8-nucleotide overhangs, generating amplicons with unique barcodes and sgRNAs. b Number of sgRNAs versus the number of plants that harbor a given sgRNA. c Number of sgRNAs inserted per plant. d Phylogenetic tree of the SlNPF1 clade showing the genes targeted by sgRNAs in the library (left) and the combinations detected using CRISPR-GuideMap in planta (right). e Bacterial population counts in 4-week-old M82 (n = 9) and 2 independent npf triple mutant plants (n = 3) at 0 and 3 DPI. Each point represents a biological replicate, averaged from three technical replicates. Statistical significance was evaluated by Students two-sided t-test, *p-value < 0.05: assuming equal variances. NS, not significant by Students t-test (p-value = 3.32 × 10-5 and 5.45 × 10−4 for alleles 1 and 2, respectively). Data are presented as box plot with the center by the median, box limits corresponding to the 25th and 75th percentiles, whiskers extending to the minimum and maximum values. f Representative images of symptoms on leaflets of M82 and two alleles of the npf triple mutant at 4 DPI. Scale bar = 1 cm. g Phylogenetic tree of the SlPT clade, showing the targeted sgRNA combinations in the library design and the combinations detected in plants. Solyc09g073010 has not been previously named and is referred to as PT9. h–j Phenotypic data of 8-week-old M82 and pt2/pt6 mutant plants grown under normal and low-phosphate conditions. h n = 13 in M82 control conditions, n = 10 in M82 low P, and pt2/pt6. i, j n = 14 in M82 control conditions, n = 10 in M82 low P, n = 10 in pt2/pt6 control conditions, n = 9 in pt2/pt6 low P. Data are presented as box plot with the center by the median, box limits correspond to the 25th and 75th percentiles, whiskers extending to the minimum and maximum values: h shoot biomass dry weight, not significant by Students two-sided t-test (p-value = 0.992) (i) root surface area, not significant by Students two-sided t-test (p-value = 0.872) and j number of root tips. Not significant by Students two-sided t-test (p-value = 0.603) Statistical significance was evaluated by Students t-test, *p-value < 0.05, **p-value < 0.01, assuming equal variances. k Representative images of the whole root system of M82 and pt2/pt6 mutant under normal (30 ppm) and low (3 ppm) phosphate conditions. Scale bar = 2 cm. Source data are provided as a Source Data file.
