Expanding the Potential of CRISPR-Cpf1-Based Genome Editing Technology in the Cyanobacterium Anabaena PCC 7120

Abstract

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.

HetF protein is a new divisome component in a filamentous and developmental cyanobacterium

“…3A). Consistent with this prediction, HetF D425 GFP fluorescence was found mostly at the periphery of the cells when overexpressed under the control of a synthetic inducible promoter (CT) (33)(34)(35) (Fig. 3C).…”

“…HetF, with an apparent molecular weight of 91.8 kD, was detected in WT but not in Δ hetF (lower panel). In all strains, HetF mutant forms was detected at levels even higher than in the WT, which is expected since these proteins were expressed from a replicative plasmid pCT (33,35). Therefore, the point mutations did not affect HetF stability.…”

“…To investigate hetF functions, a markerless deletion mutant of hetF was created as described (33) ( Fig. S1).…”

Functional Dissection of Genes Encoding DNA Polymerases Based on Conditional Mutants in the Heterocyst-Forming Cyanobacterium Anabaena PCC 7120

“…It is not clear yet how the lack of DNA polymerases or interruption of DNA replication could affect other aspects of cellular processes, such as the cell cycle and heterocyst development. In a previous study, we created a conditional mutant of polA in Anabaena PCC 7120 by replacing its ribosome binding site (RBS) with a theophylline inducible riboswitch (TRS; Niu et al, 2018). Here, we constructed another conditional mutant of dnaENI via replacing its RBS with a synthetic CT promoter that is induced by copper and theophylline.…”

Preventing Accidental Heterocyst Development in Cyanobacteria

“…One used in the study by Higo et al is the dCas9-based CRISPR interference (CRISPRi) system combined with a double control with the copper-inducible petE promoter and the anhydrotetracycline-inducible tetR promoter (26). The second tool relies on CRISPR/Cpf1-assisted gene replacement on the chromosome and the control of gene expression by a synthetic promoter under the control of theophylline alone or with copper (27). In both cases, the expression of target genes can be turned down to a sufficiently low level so that their function can be examined.…”