Figure 2.

Reliability validation of screening experiments. (A) Different sgRNA activities are related to the cellular survival rates. Mutations were introduced at different positions in the yneE-WT sgRNA N20 region (yneE-m1, yneE-m2 and yneE-m3). These sgRNA expression plasmids were transformed into the host strains expressing Cas9 and dCas9, and the survival ratios were determined by counting the colony number after overnight cultivation on agar plates. Data represents the mean ± s.d. from n = 2 biological replicates from one experiment. (B) The genome-wide sgRNA activity screenings were consistent between biological replicates. Read count of each sgRNA obtained from NGS was used to compare the agreement between biological replicates (n = 2). (C) The results from sgRNA activity screenings were highly reproducible. One additional sgRNA library (tiling library, 3451 members) was subjected to activity screening using the same protocol. The activity scores of 901 common members between this tiling library and the genome-wide sgRNA library obtained from relevant screening experiments are plotted against each other (R² = 0.771). (D) The pooled sgRNA activity screening result was confirmed by cloning 15 sgRNAs individually and measuring their activities via transformation assay (colony number counting) (as in (A)). Data represents the mean ± s.d. of biological replicates (n = 2) from one experiment. The validation result was compared with the relative abundance changes of relevant sgRNAs obtained in high-throughput profiling (R² = 0.840).