Donor plasmid library design and construction

The donor plasmid inserts can be found in Table S1. T7 (V01146) and Bas63 (MZ501086) genome files were obtained from GenBank. To design sgRNAs for T7, DeepSpCas9 and CRISPRon gRNA efficiency prediction tools were used^122,123. We find that the predicted efficiencies were concordant and opted to only use CRISPRon to design the Bas63 sgRNAs. For each sgRNA, the theoretical cut site (3nt upstream of the PAM) was used as the starting position for the design of the 95nt homology arms. The homology arms are designed such that the barcode will be inserted at the cut site position. The sgRNA, 5’ homology arm, 3’ homology arm, J23119 promoter, primer binding, and barcode landing sequences were combined with a custom Python script to create the donor plasmid insert sequences. These sequences were ordered as oligo pools (Twist Bioscience).

The barcode insert contains a 16N sequence flanked by two constant sites containing stop codons in every reading frame and is used as primer binding sites to extract barcodes for sequencing. At the termini of the insert are BsmBI or BsaI cutsites for assembly into the donor plasmid vector. BC_oligo100, which contains the 16N barcodes, is first annealed and extended with either Twist_Bcoligo100_F (BsaI termini) or Twist_Bcoligo100_BsmBI_F (BsaI termini) by adding 1μL of each oligo (100μM) to 10μL 2X KAPA HIFI Master Mix and 8μL water. This mix was incubated at 95°C for 30 seconds, 63°C for 30 seconds, and then 72°C for 30 seconds. The annealed oligos were then diluted 10-fold with the addition of 180μL of water. 1μL of the dilution was used as template with primer pairs (10uM final concentration) Twist_Bcoligo100_F + Twist_Bcoligo100_R (BsaI termini) or Twist_Bcoligo100_BsmBI_F + Twist_Bcoligo100_BsmBI_R (BsmBI termini). PCR amplification was performed as follows: 95°C 3 minutes, (98°C 20 seconds→66°C 15 seconds→72°C 15 seconds) x 20 cycles, 72°C 15 seconds, and 4°C hold. To mitigate PCR bubble formation, one additional round of PCR was performed after the addition of 1uL of each primer. Each barcode insert was gel extracted and quantified as described above.

To insert the T7 donor oligo sequences into the pUC19-Donor-BsmBI vector, a Golden Gate Assembly (BsmBI) was performed using 75ng of pUC19-Donor-BsmBI and a 2-fold molar ratio of oligo insert. For Bas63 donor oligo sequences, pUC19-Donor-BsaI vector was used with Golden Gate Assembly (BsaI) Mix. The reactions were performed as described above. The reactions were dialyzed and transformed as describe above. Recovery was performed in SOC for 30 minutes. 100μL of the recovered transformants was plated on LB-Kan plates and the rest was used to inoculate 5mL of LB-Kan media for overnight the growth. Plasmid DNA from the resulting outgrowth was extracted as described above. The resulting purified plasmids pUC19- T7Library and pUC19-Bas63Library are the landing vectors for the barcode insert.

To insert the barcode insert (BsaI termini) into pUC19-T7Library, a Golden Gate Assembly (BsaI) was performed using 75ng of pUC19-T7Library and a 2-fold molar ratio of barcode insert. For barcode insert (BsmBI termini), pUC19-Bas63Library vector was used with Golden Gate Assembly (BsmBI) Mix. The dialysis, transformation, and recovery outgrowth were handled the same as during the donor oligo sequence insertion. Serial dilutions of the recovered transformants were plated on LB-Kan plates and used to inoculate 5mL of LB-Kan media for overnight the growth. We proceeded with the dilution that gave approximately 10-fold coverage of the theoretical library size. Plasmid DNA from the resulting outgrowth was extracted as described above. The resulting plasmid libraries are pUC19-T7LibraryBC and pUC19-Bas63LibraryBC.

E. coli DH10B Cas9-RecA competent cells were made the same way as described with E. coli DH10B with three differences. Cultures were scaled down to 10mL, all growth steps occurred at 30°C, and the LB media used was supplemented with spectinomycin. 1ng of pUC19-T7LibraryBC and pUC19-Bas63LibraryBC was transformed into 25μL of Cas9-RecA competent cells. Transformants were recovered at 30°C for 30 minutes. Serial dilutions of the recovered transformants were plated on LB-Kan-Spec plates and used to inoculate 5mL of LB-Kan-Spec media for overnight the growth. We proceeded with the dilution that gave approximately 10-fold coverage of the theoretical library size. Glycerol stocks for both libraries were made and stored at 80°C. Plasmid DNA from the resulting outgrowth was extracted as described above.

To generate sequencing amplicons for both libraries, 1ng of plasmid DNA was used as template for a 10μL PCR reaction with primer pairs DelLib{2,3,4}N_NGS_F + DelLib{2,3,4}N_NGS_R containing 2, 3, and 4 N offsets and the following PCR cycle settings: 95°C 5 minutes, (98°C 20 seconds→65°C 15 seconds→72°C 15 seconds) x 15 cycles, 72°C 1 minute, and 4°C hold. Illumina i7 and i5 adapter sequences containing unique indices and were added after performing a second 25μL PCR reaction with the following cycle settings: 95°C 3 minutes, (98°C 20 seconds→65°C 15 seconds→72°C 15 seconds) x 10 cycles, 72°C 1 minute, and 4°C hold. PCR products were purified with the the E.Z.N.A Cycle Pure Kit and quantified using the Qubit 4 as described above. The purified products were pooled and sequenced on an Illumina MiSeq with a 2 x 250 Miseq Reagent v2 kit.

Read pairs were merged and filtered with Fastp v0.23.4¹²⁴, requiring a minimum of 90% of the read having scores above Q25. The adapter sequences were removed, and relevant donor sequences were extracted using Cutadapt v4.1¹²⁵. The resulting sequences were further analyzed using custom Python scripts. The result is the mapping of barcodes to their theoretical insertion site (genic or intergenic regions).