Transformation compared to TARDIS.

For transformation, a large population of cells are individually transformed with a DNA library, resulting in a diverse population of individuals. TARDIS achieves a diversity of individuals by splitting transgenesis into two separate processes: (1) The introduction of a diverse library, which is formed into a TARDIS library array, passed down to future generations and thus replicated; and (2) an event that triggers the integration a sequence from the library at random, resulting in a diversity of integrated sequences.

Barcode landing pad and diverse donor library.

A) Schematic design for the barcode landing pad and integration. A broken hygromycin resistance gene is targeted by Cas9, which repairs off the TARDIS array, integrating a barcode and restoring the functionality of the gene. B) The TARDIS multiplex library was created from a randomized oligo library, which underwent 10 cycles of PCR to make a dsDNA template. The barcode fragment was then added into a three fragment overlap PCR to add homology arms and make the final library for injection.

TARDIS Library Arrays can contain large barcode diversity.

A) Frequency distribution of 1,319 unique barcodes in array 1 (PX816). B) Frequency distribution of the 3,001 unique barcode sequences in array 2 (PX817). C) Sequence logo probabilities of the 15 base pair positions of the barcodes in the injection mix, array 1 and array 2.

Integration frequency from TARDIS Library Array to F1.

A) Frequency of integration from TARDIS Library Array to the F1, R ≈ 0.96, p ≈ 5.7×10−154. Different colors represent four biological replicates. Line shading represents 95% confidence interval. B) Sequence probabilities of PX786 compared to the F1 integrations.

TARDIS promoter library.

A) Overview of the two split landing pads and their associated promoter insertion vectors. Both the selective marker and the fluorophore expression are restored upon correct integration. B) Transcriptional reporters for nine genes were recovered from a single heatshock of a single TARDIS array line (PX819). Integration was into the single mScarlet/HygR landing pad. Main images show mScarlet-I expression for the indicated reporter while insets show polarized image of the same region. C) Example simultaneous, dual integration from a single TARDIS array into the double landing pad strain with PEST. ceh-10p::mNeonGreen::PEST is false colored green and ceh-40p::mScarlet-I::PEST is false colored magenta. All scale bars represent 20μm

Characteristics of injected promoters and presence in tested array line (PX819) and integrated lines derived from that array.

Key Reagents.

supplements/514301_file02.pdf Supplemental Document 1supplements/514301_file03.xlsx Supplemental Table 4supplements/514301_file04.xlsx Supplemental Table 2supplements/514301_file05.xlsx Supplemental Table 1supplements/514301_file06.xlsx Supplemental Table 3

Schematic layout for the two separate PCR processes for identification of barcode counts in arrays (Amplicon One-Array) and integrants (Amplicon One-Integrant).

A) Barcode frequencies for Injection mix used for arrays 1, 2, and 3. There are approximately 1 million reads represented. In total, 797353 unique sequences were identified. A few of these unique sequences were represented at a higher frequency with a count cutoff greater than 50.

TARDIS Array 3. Two individual arrays were isolated from the same plate. Both show considerably less diversity than TARDIS arrays 1 and 2. Distribution of unique barcode frequencies, sequence logo base pair probability, and count cut off for A) TARDIS Array 3 profile 1 and B) TARDIS Array 3 profile 2.

Determination of proper count cutoff for A) TARDIS Array 1 and B) TARDIS Array 2.

F1 integration events followed a consistent pattern, with replicated outlier barcode sequences. Generally, the same barcodes integrated at approximately the same frequency across the four replicates.

(A) Promoters from two array bearing lines were amplified using universal primers and show two distinct profiles. Note that not all promoters found in the arrays were detected in this screen. (B) Line PX819 was heatshocked to trigger integration and four hygromycin resistant progeny were singled from each of the 59/60 plates with hygromycin resistant individuals. Singled worms were screened by PCR and select promoters chosen for sequencing based on their size profile. PCR and sequencing result are shown for representative plates. *=Due to their size of the egl-46 and nhr-67 promoters do not reliably amplify with the universal primers. Therefore, samples with no or weak amplification were rescreened with primers specific to these two promoters (not shown).