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.
Keywords: CRISPR; genetic barcoding; genome editing; insertion sites; neutral sites; synthetic biology.