Figure 3.
A library screen confirms differential depletion for some AsCas12a guides across strains and sets guide design rules for efficient antimicrobial activity. (A) Schematic of the library screen setup. (B) Depletion of guides following nuclease induction for 3 h or overnight. (C) Box plots of the guide depletion distribution before and after aTc nuclease induction for the targeting and NT guides. MFDpir: E. coli donor strain. KPPR1, SB5442 and NTUH-K2044 (indicated as NTUH): different Kp strains. T0, T3h, TON: time 0, 3 h, overnight after induction. (D) Library verification with five different gRNAs. After transforming the library into E. coli, five colonies were randomly selected, transformed into different Kp strains and induced to determine the reduction in colony numbers. Subsequently, they were sequenced to determine the expressed gRNA sequence and are reported in the initial assigned order. KP1_0269 indicates a gene encoding a putative protein, while xseA-guaB and yebK-KP1_3497 are placed in intergenic regions. The gRNA sequences can be found in Supplementary Table S1. *: small colonies on plates. (E) Depiction of the method used for developing the machine learning algorithm. (F) Contribution of different features to guide depletion from the library using the mixed dataset for training the algorithm. The left barplot depicts the absolute weight of each feature to the depletion values, while the right beeswarm plot shows how each datapoint impacts depletion according to each feature. The PAM nucleotides are counted from 3′ to 5′ starting from the −1 position before the gRNA sequence. (G) Correlation between guide depletion in the KPPR1 and NTUH-K2044 strains. (H) Validation of algorithm prediction of high and low-efficiency guides in KPPR1 and SB5442. The dashed line represents the limit of detection of the assay. Each dot represents a biological replicate obtained by preparing electrocompetent cells from a separate colony.