Nanopore sequencing of RNA guided transposition in cyanobacteria
In genome engineering, integration of incoming DNA has been dependent on enzymes produced by dividing cells which has been a bottle neck towards increasing DNA-insertion frequencies and accuracy. RNA-guided transposition with CRISPR-associated transposase (CAST) was shown to be highly effective and specific in Escherichia coli. Here we developed Golden-Gate vectors to test this approach in filamentous cyanobacteria and show that CAST is effective in Anabaena sp. strain PCC 7120. The comparatively large plasmids containing the CAST and the engineered transposon were successfully transferred into Anabaena via conjugation using either suicide or replicative plasmids. Single guide RNA that target the leading, but not the reverse complement strand were effective with the protospacer associated motif (PAM) sequence included in the single guide RNA. In four out of six cases analyzed over two distinct target loci, the insertion site was exactly 63 bases after the PAM. CAST on a replicating plasmid was toxic which could be used to cure the plasmid. In all six cases analyzed, only the transposon defined by the sequence ranging from left and right elements was inserted at the target loci, therefore, RNA-guided transposition resulted from cut and paste. No endogenous transposons were remobilized by exposure to CAST enzymes. This work is foundational for genome editing by RNA-guided transposition in filamentous cyanobacteria, whether in culture or in complex communities.