Meng Wei, Qiao Kai, Liu Fan, Gao Xu, Hu Xuan, Liu Jia, Gao Yiyun, Zhu Jianrong
School of Environment, Beijing Normal University, Beijing, China.
R & D Centre of Aerobic Granule Technology, Beijing, China.
Biotechnol J. 2023 Sep;18(9):e2200596. doi: 10.1002/biot.202200596. Epub 2023 Jun 17.
Aerobic granular sludge (AGS) is a microbial aggregate with a biofilm structure. Thus, investigating AGS in the aspect of biofilm and microbial attachment at the genetic level would help to reveal the mechanism of granule biofilm formation. In this work, a two-plasmid clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas)12a genome editing system was constructed to identify attachment genes for the first time in Stenotrophomonas AGS-1 from AGS. One plasmid contained a Cas12a cassette driven by an arabinose-inducible promoter, and another contained the specific crRNA and homologous arms (HAs). Acidaminococcus sp. Cas12a (AsCas12a) was adopted and proven to have mild toxicity (compared to Cas9) and strong cleavage activity for AGS-1. CRISPR/Cas12a-mediated rmlA knockout decreased attachment ability by 38.26%. Overexpression of rmlA in AGS-1 resulted in an increase of 30.33% in attachment ability. These results showed that the modulation of rmlA was an important factor for the biofilm formation of AGS-1. Moreover, two other genes (xanB and rpfF) were knocked out by CRISPR/Cas12a and identified as attachment-related genes in AGS-1. Also, this system could achieve point mutations. These data indicated that the CRISPR/Cas12a system could be an effective molecular platform for attachment gene function identification, which would be useful for the development of AGS in wastewater treatment.
好氧颗粒污泥（AGS）是一种具有生物膜结构的微生物聚集体。因此，从基因水平研究AGS在生物膜和微生物附着方面的情况，将有助于揭示颗粒生物膜形成的机制。在这项工作中，构建了一个双质粒成簇规律间隔短回文重复序列（CRISPR）/CRISPR相关蛋白（Cas）12a基因组编辑系统，首次在AGS的嗜麦芽窄食单胞菌AGS-1中鉴定附着基因。一个质粒包含由阿拉伯糖诱导型启动子驱动的Cas12a盒，另一个包含特异性crRNA和同源臂（HAs）。采用嗜酸栖热菌Cas12a（AsCas12a），并证明其对AGS-1具有轻度毒性（与Cas9相比）和较强的切割活性。CRISPR/Cas12a介导的rmlA基因敲除使附着能力降低了38.26%。在AGS-1中过表达rmlA导致附着能力提高了30.33%。这些结果表明，rmlA的调控是AGS-1生物膜形成的一个重要因素。此外，另外两个基因（xanB和rpfF）被CRISPR/Cas12a敲除，并被鉴定为AGS-1中的附着相关基因。而且，该系统可以实现点突变。这些数据表明，CRISPR/Cas12a系统可能是一个用于附着基因功能鉴定的有效分子平台，这将有助于AGS在废水处理中的应用发展。
