CRISPR systems, such as CRISPR-Cas9 and CRISPR-Cpf1, have been successfully used for genome editing in a variety of organisms. Although the technique of CRISPR-Cpf1 has been applied in cyanobacteria recently, its use was limited without exploiting the full potential of such a powerful genetic system. Using the cyanobacterium Anabaena PCC 7120 as a model strain, we improved the tools and designed genetic strategies based on CRISPR-Cpf1, which enabled us to realize genetic experiments that have been so far difficult to do in cyanobacteria. The development includes: (1) a “two-spacers” strategy for single genomic modification, with a success rate close to 100%; (2) rapid multiple genome editing using editing plasmids with different resistance markers; (3) using sacB, a counter-selection marker conferring sucrose sensitivity, to enable the active loss of the editing plasmids and facilitate multiple rounds of genetic modification or phenotypic analysis; (4) manipulation of essential genes by the creation of conditional mutants, using as example, polA encoding the DNA polymerase I essential for DNA replication and repair; (5) large DNA fragment deletion, up to 118 kb, from the Anabaena chromosome, corresponding to the largest bacterial chromosomal region removed with CRISPR systems so far. The genome editing vectors and the strategies developed here will expand our ability to study and engineer cyanobacteria, which are extensively used for fundamental studies, biotechnological applications including biofuel production, and synthetic biology research. The vectors developed here have a broad host range, and could be readily used for genetic modification in other microorganisms.

The filamentous cyanobacterium Anabaena sp. PCC 7120 develops N 2-fixing heterocyst cells under condition of combined-nitrogen deprivation and constitutes an excellent model for studying cell differentiation. The mechanism of heterocyst development has been extensively investigated and a network of regulating factors has been identified. A few studies have showed that the process of heterocyst differentiation relates with cell cycle events, but further investigation is still required to understand this relationship. In a previous study, we created a conditional mutant of PolI encoding gene, polA, by using a CRISPR/Cpf1 gene-editing technique. Here, we were able to create another conditional mutant of a PolIII encoding gene dnaENI using a similar strategy and subsequently confirmed the essential roles of both polA and dnaENI in DNA replication. Further investigation on the phenotype of the mutants showed that lack of PolI caused defects in chromosome segregation and cell division, while lack of DnaENI (PolIII) prevented bulk DNA synthesis, causing significant loss of DNA content. Our findings also suggested the possible existence of a SOS-response like mechanism operating in Anabaena PCC 7120. Moreover, we found that heterocyst development was differently affected in the two conditional mutants, with double heterocysts/proheterocysts found in PolI conditional mutant. We further showed that formation of such double heterocysts/proheterocysts are likely caused by the difficulty in nucleoids segregation, resulting delayed, or non-complete closure of the septum between the two daughter cells. This study uncovers a link between DNA replication process and heterocyst differentiation, paving the way for further studies on the relationship between cell cycle and cell development.