Parse Bacillus methanolicus genome and native plasmids for PAM sequence in upstream half of all features. Add 20 bps sequence upstream of hits to sgRNA list. Drop sgRNAs with off-target hits >0 of query sequence with 0 & 1 mismatches and off-target hits >10 of query sequence with 2 mismatches.Vary query sequence and mutation sequence (2bp mismatches) to generate 3 libraries: Downstream 13 bps query & mutation sequence resulting in library 0, 20 bp query and downstream 13 bps mutation sequence resulting in library 1 and the whole 20 bps query & mutation sequence resulting in library 2.
Generated libaries are included as csv files.