2 research outputs found
Predicted ΔG melting energies are shown for each putative hairpin structure
Conserved sequence R5 (csR5) is identical to the previously described LTRR in sp. strain PCC 7120 [24].<p><b>Copyright information:</b></p><p>Taken from "Transcription of the extended -operon in sp. strain PCC 7120"</p><p>http://www.biomedcentral.com/1471-2180/8/69</p><p>BMC Microbiology 2008;8():69-69.</p><p>Published online 28 Apr 2008</p><p>PMCID:PMC2408588.</p><p></p
Genome Engineering by RNA-Guided Transposition for <i>Anabaena</i> sp. PCC 7120
In genome engineering, the integration of incoming DNA
has been
dependent on enzymes produced by dividing cells, which has been a
bottleneck toward 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 to show that
it is effective in Anabaena sp. strain PCC 7120.
The comparatively large plasmids containing 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