CRISPR/Cas systems in cyanobacteria, their involvement in cell differentiation and potential for metabolic manipulation

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CRISPR interference (CRISPRi) is currently the most popular technology for the manipulation of gene expression and genome editing. In most of these approaches, the CRISPR-Cas Type II enzyme Cas9 is used together with a guide RNA to target specific regions in the chromosomal DNA. These Class 2 CRISPR-Cas systems are supposed to have originated from transposable elements that are particularly diverse and abundant in cyanobacteria. Biocomputational analyses suggest that certain filamentous cyanobacteria are particularly rich, with possibly 11 CRISPR-Cas systems in the model organism Anabaena sp. PCC7120. These species differentiate heterocysts, specialized cells for nitrogen fixation, following a series of genetic switches. Here we suggest to analyse the CRISPR-Cas systems in Anabaena sp. PCC7120 and to investigate a possible link to heterocyst differentiation. We address a subtype III-D system that is inserted in the genetic element interrupting the fdxN gene, becomes excised during cell differentiation, uses an unusual Cas1-reverse transcriptase fusion protein and a bipartite repeat-spacer array. We will characterize the novel Class 2 c2c5 CRISPR-Cas system that has been predicted to rely on Alr3613 as effector protein, using biochemical and molecular-genetic approaches. It is not understood how recently discovered Class 2 systems lacking cas1 genes can adapt their repeat-spacer arrays to new invaders. We address this question assuming that these either depend on one of the three classical systems in Anabaena sp. PCC7120 that provide Cas1 activity in trans or utilize the recombinases involved in heterocyst differentiation using interference assays and mutant analyses. This work will be complemented by global approaches targeting the functionality of all 11 possible systems. These analyses will lead to the characterization of novel CRISPR-Cas effector proteins that will complement the existing suite of Cas9, C2c1 and Cpf1 proteins, foster the construction of efficient and genetically stable cyanobacterial producer strains for the biotechnology of biofuels and other beneficial metabolites and provide substantial new insight into the diverse functionalities of CRISPR-Cas systems and the differentiation of bacterial cells.