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与指导RNA一起使用以靶向染色体DNA中的特定区域。这些2类CRISPR-Cas系统被认为起源于在蓝藻中特别多样和丰富的转座因子。生物计算分析表明，某些丝状蓝藻特别丰富，在模式生物鱼腥藻PCC 7120中可能有11个CRISPR-Cas系统。这些物种分化异形胞，专门用于固氮的细胞，遵循一系列遗传开关。在这里，我们建议分析鱼腥藻PCC 7120中的CRISPR-Cas系统，并研究与异形胞分化的可能联系。我们解决了一个亚型III-D系统，该系统插入到中断fdxN基因的遗传元件中，在细胞分化过程中被切除，使用了一种不寻常的Cas 1-逆转录酶融合蛋白和一个二分重复间隔区阵列。我们将使用生物化学和分子遗传学方法表征新型2类c2 c5 CRISPR-Cas系统，该系统已被预测依赖于Alr 3613作为效应蛋白。目前还不清楚最近发现的缺乏cas 1基因的2类系统如何使其重复间隔区阵列适应新的入侵者。我们解决这个问题的假设是，这些要么依赖于鱼腥藻PCC 7120中提供反式Cas 1活性的三个经典系统之一，要么利用干扰测定和突变体分析利用参与异形胞分化的重组酶。这项工作将得到针对所有11个可能系统的功能的全球办法的补充。这些分析将导致新型CRISPR-Cas效应蛋白的表征，这些蛋白将补充现有的Cas9，C2 c1和Cpf 1蛋白，促进构建用于生物燃料和其他有益代谢物生物技术的高效和遗传稳定的蓝藻生产菌株，并为CRISPR-Cas系统的多种功能和细菌细胞的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.