Fig. 2: Expanding TIGER for extended target selection, SNP detection and quantitative recording.
From: RNA recording in single bacterial cells using reprogrammed tracrRNAs
a, Using DNA targets lacking a C in the editing window. A representative guide derived from the CJ8421_04975 mRNA was paired with CJ Rptr3 along with the corresponding DNA target. At different locations within the target, a C was swapped into the target along with a preceding favored T or disfavored A (if not already present). The guide portion of the CJ8421_04975 transcript was mutated so there are no mismatches (match) or a single mismatch (mismatch) at the introduced C. b, Bulk sequencing of the different guide–DNA target pairs (Fig. 1e). c, Enhancing SNP detection through the use of helper mutations. The SNP (pink) was swapped into position 19 of the guide portion of the CJ8421_04975 transcript. A helper mutation (orange) was also added at position 18 of the guide. d, Bulk sequencing of CJ8421_04975 transcript recording with the different guide–target mutations. The swapped bases are within the gray box. e, Recording the constitutively expressed degfp transcript. f, Bulk sequencing of degfp transcript recording when placing the TIGER machinery on plasmids with different copy numbers. H, high-copy; M, medium-copy and L, low-copy. g, Assessing quantitative recording by varying the promoter strength of degfp. h, Correlating promoter strength and recording efficiency. The left shows bulk sequencing. GFP fluorescence was measured by flow cytometry analysis. See g for the corresponding colors. The P value was calculated using linear regression analysis, n = 12. The right shows colony sequencing and single-cell sequencing from one culture with the strongest J23119 promoter. a.u., arbitrary units. Values in b, d and f represent the mean and standard deviation of independent experiments starting from three separate colonies. Each dot for colony sequencing or single-cell DNA sequencing represents one of 20 sequenced colonies or single cells in the biological replicate. The dot plot is representative of independent experiments starting from three separate colonies. See the representative plots and gates associated with single-cell DNA sequencing in Extended Data Fig. 2b. *P < 0.05, **P < 0.01, ***P < 0.001, NS (not significant) P > 0.05. P values were calculated using a two-tailed Student’s t-test with unequal variance.
