Fig. 5

Assembly of level 1 polycistronic tRNA-sgRNA expression modules to be used with SpCas9. During the first step (a) annealed complementary oligos encoding the guide are inserted into a level 0 acceptor with the tRNA-sgRNA backbone using BpiI. During the second step (b), tRNA-sgRNA modules are fused with the respective Pol III promoter using BsaI. Assembly of less than six tRNA-sgRNA modules into a level 1 vector requires respective endlinkers (c). pFH and pAK constructs shown in black font carry the improved sgRNA backbone [33], while the ones shown in blue font – the classic sgRNA backbone [34]. *This is a published module [35]