Abstract
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. Recently, RNA-guided transposition with CRISPR-associated transposase (CAST) was reported as highly effective and specific in Escherichia coli. Here we developed Golden-Gate vectors to test CAST in filamentous cyanobacteria and show that it 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(sg)-RNA encoding the leading, but not the reverse complement strand of the target were effective with the protospacer associated motif (PAM) sequence included in the sgRNA. 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 cargo 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.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
We now incorporated the GFP fluorescence in Figure 4, included documentation of the Indels listed in Table S3 in supporting figures. We furthermore have amended the methods, included the sequence accessions, and we hope improved the clarity of the entire manuscript. We further altered the title to represent the results. The application of the method to symbiotic cyanobacteria is the subject of our next paper.
