Abstract
Cyanobacteria are the ideal organisms for the production of a wide range of bioproducts as they can convert CO(2) directly into the desired end product using solar energy. Unfortunately, the engineering of cyanobacteria to create efficient cell factories has been impaired by the cumbersome genetic tools that are currently available for these organisms; especially when trying to accumulate multiple modifications. We sought to construct an efficient and precise tool for generating numerous markerless modifications in cyanobacteria using CRISPR technology and the alternative nuclease, Cpf1. In this study we demonstrate rapid engineering of markerless knock-ins, knock-outs and point mutations in each of three model cyanobacteria; Synechococcus, Synechocystis and Anabaena. The markerless nature of cpf1 genome editing will allow for complex genome modification that was not possible with previously existing technology while facilitating the development of cyanobacteria as highly modified biofactories.