CRISPR Interference for Rapid Knockdown of Essential Cell Cycle Genes inLactobacillus plantarum

Abstract

Studies of essential genes in bacteria are often hampered by the lack of accessible genetic tools. This is also the case for Lactobacillus plantarum, a key species in food and health applications. Here, we develop a clustered regularly interspaced short palindromic repeat interference (CRISPRi) system for knockdown of gene expression in L. plantarum. The two-plasmid CRISPRi system, in which a nuclease-inactivated Cas9 (dCas9) and a gene-specific single guide RNA (sgRNA) are expressed on separate plasmids, allows efficient knockdown of expression of any gene of interest. We utilized the CRISPRi system to gain initial insights into the functions of key cell cycle genes in L. plantarum. As a proof of concept, we investigated the phenotypes resulting from knockdowns of the cell wall hydrolase-encoding acm2 gene and of the DNA replication initiator gene dnaA and of ezrA, which encodes an early cell division protein. Furthermore, we studied the phenotypes of three cell division genes which have recently been functionally characterized in ovococcal bacteria but whose functions have not yet been investigated in rod-shaped bacteria. We show that the transmembrane CozE proteins do not seem to play any major role in cell division in L. plantarum. On the other hand, RNA-binding proteins KhpA and EloR are critical for proper cell elongation in this bacterium. IMPORTANCE L. plantarum is an important bacterium for applications in food and health. Deep insights into the biology and physiology of this species are therefore necessary for further strain optimization and exploitation; however, the functions of essential genes in the bacterium are mainly unknown due to the lack of accessible genetic tools. The CRISPRi system developed here is ideal to quickly screen for phenotypes of both essential and nonessential genes. Our initial insights into the function of some key cell cycle genes represent the first step toward understanding the cell cycle in this bacterium.

Comparative CRISPR type III-based knockdown of essential genes in hyperthermophilic Sulfolobales and the evasion of lethal gene silencing

“…Similar trends were reported for silencing assays using an antisense RNA approach in E. coli, where 65% silencing of ftsZ, encoding the central cell division protein was achieved which led to 50% reduction of the growth rate [54]. Furthermore, heterologous expression of the catalytically inactive CRISPR-dCas9 enzyme, was used to inhibit transcription of essential genes in other bacterial genera, such as Lactobacillus [24], Staphylococcus [55] or Mycobacterium [22], which again all showed strong growth retardation and a reduction in viability upon maximum silencing. Thus, the phenotypes observed in our survey of a hyperthermophilic archaeon are similar to findings in other mesophilic organisms where different silencing approaches were used to target essential genes.…”

“…For essential gene studies in eukaryotes and bacteria, mainly nuclease-inactive derivatives of DNA-targeting CRISPR type II and V proteins (i.e. dCas9 and ddCas12a) have been employed that, when expressed in trans, bind to the respective promoter or coding region to block transcription [22][23][24][25][26]. Similarly, a mutated variant of the DNA-targeting type I protein complex (CASCADE) deficient in Cas3 nuclease activity, was successfully harnessed to silence essential genes in a halophilic archaeon [18].…”

Endogenous membrane stress induces T6SS activity in Pseudomonas aeruginosa

“…By directing dCas9 to the promoter region or 3′ end of a gene with a single guide RNA (sgRNA), the CRISPRi system can be used to transcriptionally control target gene expression ( 66 , 67 ). CRISPRi has been established in several organisms such as Escherichia coli , Lactobacillus plantarum , Bacillus subtilis , Mycobacterium tuberculosis , Streptococcus pneumoniae , and V. cholerae and has been shown to be a useful tool to study essential genes ( 67 – 73 ). The CRISPRi technology is applicable to high-throughput screens as well and has other advantages particularly in the study of essential gene products compared to conventional knockdown methods ( 67 ).…”

Perspectives for Glyco-Engineering of Recombinant Biopharmaceuticals from Microalgae

“…Similar to exogenous enzyme insertion, endogenous enzyme removal strategies can be random or targeted. Gene(s) knock-down (KD) strategies result in reduced activity of the glycosylation enzymes and can be achieved by gene(s) silencing techniques, such as RNA interference [162] or CRISPR interference (CRISPRi) [163]. However, gene(s) KD does not completely inactivate enzyme expression, often resulting in still high levels of the targeted undesired enzyme activity and consequently in the attachment of the unwanted immunogenic residues.…”