Gene silencing based on RNA-guided catalytically inactive Cas9 (dCas9): a new tool for genetic engineering in Leptospira

Abstract

Leptospirosis is a worldwide zoonosis caused by pathogenic bacteria of the genus Leptospira, which also includes free-living saprophyte strains. Many aspects of leptospiral basic biology and virulence mechanisms remain unexplored mainly due to the lack of effective genetic tools available for these bacteria. Recently, the type II CRISPR/Cas system from Streptococcus pyogenes has been widely used as an efficient genome engineering tool in bacteria by inducing double-strand breaks (DSBs) in the desired genomic targets caused by an RNA-guided DNA endonuclease called Cas9, and the DSB repair associated machinery. In the present work, plasmids expressing heterologous S. pyogenes Cas9 in L. biflexa cells were generated, and the enzyme could be expressed with no apparent toxicity to leptospiral cells. However, L. biflexa cells were unable to repair RNA-guided Cas9-induced DSBs. Thus, we used a catalytically dead Cas9 (dCas9) to obtain gene silencing rather than disruption, in a strategy called CRISPR interference (CRISPRi). We demonstrated complete gene silencing in L. biflexa cells when both dCas9 and single-guide RNA (sgRNA) targeting the coding strand of the β-galactosidase gene were expressed simultaneously. Furthermore, when the system was applied for silencing the dnaK gene, no colonies were recovered, indicating that DnaK protein is essential in Leptospira. In addition, flagellar motor switch FliG gene silencing resulted in reduced bacterial motility. To the best of our knowledge, this is the first work applying the CRISPRi system in Leptospira and spirochetes in general, expanding the tools available for understanding leptospiral biology.

Heterologous Expression of the Pathogen-Specific LIC11711 Gene in the Saprophyte L. biflexa Increases Bacterial Binding to Laminin and Plasminogen

“…Transcription of lic11711 under P32 promoter ( L. biflexa -LIC11711) was ~580x higher than with its native promoter (wild-type L. interrogans strain M20). The functionality of P32 in the surrogate L. biflexa is in accordance with previous works [ 14 , 19 ]. Motility or morphological differences between wild L. biflexa , L. biflexa -pMaOri and L. biflexa -LIC11711 were not observed.…”

“…Recently, the genetic tools to understand the role of leptospiral proteins have expanded, allowing heterologous expression of proteins of interest by E. coli-Leptospira shuttle vectors [ 9 , 10 ] and subsequent evaluation of gain of function phenotype. Moreover, gene knockout by random transposon [ 11 , 12 ] or homologous recombination [ 13 ], and more recently gene silencing using CRISPRi [ 14 ], have made it possible to study the loss of function phenotypes.…”

“…Here, we examined the gain-of-function approach by genetically fusing the lic11711 gene with the lipL32 promoter (P32) to overexpress the protein in L. biflexa serovar Patoc. The P32 is a strong promoter used to express proteins in heterologous systems [ 14 , 19 ]. In addition, LipL32 is a major lipoprotein of pathogenic Leptospira , expressed at high levels both during cultivation and during infection, with a putative role during mammalian infection [ 26 ].…”

Oncolytic Adenoviruses: Strategies for Improved Targeting and Specificity

“…This may impart undesirable and irreversible damages to genome. However, other CRIPR based tools like, modified CRISPR-based base editors, suppressors and enhancers (which do not cleave the target) could be safer alternatives [248][249][250][255][256][257][258].…”

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

“…Because the endogenous locus remains intact, in both cases, gene expression tends to be reduced but is not abolished, allowing survival. Besides the wellestablished RNAi (RNA interference) pathway that has been widely used to silence genes in virtually every eukaryotic model [10], new fruitful knockdown tools have recently emerged from the prokaryotic virus-defence system CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) [11][12][13][14][15][16][17][18]. CRISPR systems are widespread in bacteria and archaea and function to protect the host cell from viral infection [19,20].…”