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

CRISPR干扰（CRISPRi）是目前最流行的基因表达操纵和基因组编辑技术。在大多数这些方法中，CRISPR-Cas II型酶Cas9与引导RNA一起用于靶向染色体DNA中的特定区域。这些2类CRISPR-Cas系统被认为起源于蓝藻中特别多样化和丰富的转座元件。生物计算分析表明，某些丝状蓝藻特别丰富，在模式生物Anabaena sp. PCC7120中可能有11个CRISPR-Cas系统。这些物种分化为异囊细胞，专门用于固氮的细胞，遵循一系列的遗传开关。在这里，我们建议分析Anabaena sp. PCC7120中的CRISPR-Cas系统，并研究其与异囊分化的可能联系。我们研究了一种亚型III-D系统，该系统插入打断fdxN基因的遗传元件中，在细胞分化过程中被切除，使用一种不寻常的cas1逆转录酶融合蛋白和一种双部重复间隔器阵列。我们将利用生物化学和分子遗传学方法描述新型2类c2c5 CRISPR-Cas系统，该系统被预测依赖于Alr3613作为效应蛋白。目前尚不清楚最近发现的缺乏cas1基因的2类系统如何使它们的重复间隔阵列适应新的入侵者。我们假设这些依赖于Anabaena sp. PCC7120中的三个经典系统之一，这些系统在反式中提供Cas1活性，或者使用干扰试验和突变分析利用参与异囊分化的重组酶来解决这个问题。这项工作将以针对所有11个可能系统的功能的全球方法为补充。这些分析将导致新的CRISPR-Cas效应蛋白的表征，这些蛋白将补充现有的Cas9， C2c1和Cpf1蛋白，促进构建高效且遗传稳定的蓝藻生产菌株，用于生物燃料和其他有益代谢物的生物技术，并为CRISPR-Cas系统的多种功能和细菌细胞分化提供实质性的新见解。

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.