Putative Phenotypically Neutral Genomic Insertion Points in Prokaryotes

The barriers to effective
genome editing in diverse prokaryotic
organisms have been falling at an accelerated rate. As editing becomes
easier in more organisms, quickly identifying genomic locations to
insert new genetic functions without disrupting organism fitness becomes
increasingly useful. When the insertion is noncoding DNA for applications
such as information storage or barcoding, a neutral insertion point
can be especially important. Here we describe an approach to identify
putatively neutral insertion sites in prokaryotes. An algorithm (targetFinder)
finds convergently transcribed genes with gap sizes within a specified
range, and looks for annotations within the gaps. We report putative
editing targets for 10 common synthetic biology chassis organisms,
including coverage of available RNA-seq data, and provide software
to apply to others. We further experimentally evaluate the neutrality
of six identified targets in Escherichia coli through
insertion of a DNA barcode. We anticipate this information and the
accompanying tool will prove useful for synthetic biologists seeking
neutral insertion points for genome editing.