Induced protein expression in Leptospira spp. and its application to CRISPR/Cas9 mutant generation

Authors: FERNANDES, LUIS - Oak Ridge Institute For Science And Education (ORISE); NASCIMENTO, ANNA - Butantan Institute; Nally, Jarlath

Submitted to: Scientific Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/29/2025
Publication Date: 2/5/2025
Citation: Fernandes, L.G., Nascimento, A.L., Nally, J.E. 2025. Induced protein expression in Leptospira spp. and its application to CRISPR/Cas9 mutant generation. Scientific Reports. https://doi.org/10.1038/s41598-025-88633-w.
DOI: https://doi.org/10.1038/s41598-025-88633-w

Interpretive Summary: For many years, genetic manipulation of Leptospira spp. has been a labor-intensive and challenging process, with most studies relying on random transposon libraries. The introduction of the prokaryotic “adaptive immune” CRISPR/Cas9 system has revolutionized the field, enabling targeted gene silencing and knockouts in various Leptospira species and serovars. Gene silencing has been achieved using CRISPR interference (CRISPRi), which employs the constitutive expression of a catalytically inactive Cas9 (dCas9) and a single-guide RNA (sgRNA). This nucleoprotein complex binds to the target gene, guided by base pairing, and blocks transcription to achieve silencing. However, the delivery of plasmids via E. coli-mediated conjugation can sometimes fail, potentially leading to misinterpretation of gene essentiality. To address this, we developed a cassette for controlled protein expression in Leptospira, utilizing the lac operon system, where protein expression is induced by IPTG. After validating this system, we replaced the constitutive promoter in CRISPRi plasmids with our IPTG-inducible cassette, enabling controlled dCas9 expression. This allowed us to perform conjugation experiments on agar plates with and without IPTG, providing a more robust assessment of gene essentiality by distinguishing failed conjugation events from lethality due to gene silencing. Furthermore, the cassette was adapted for controlled Cas9 expression, enabling the introduction of double-stranded DNA breaks in the genome. These breaks are repaired by a constitutively expressed non-homologous end-joining (NHEJ) system, resulting in mutations. This novel toolkit enhances genetic manipulation of Leptospira spp., paving the way for deeper insights into leptospiral biology and virulence. Additionally, it facilitates the generation of knockout mutants for use in virulence studies and the development of improved veterinary vaccines.

Technical Abstract: Expanding the genetic toolkit for Leptospira spp. is a crucial step toward advancing our understanding of the biology and virulence of these atypical bacteria, and ultimately facilitating the development of improved vaccines and diagnostic methods. Pathogenic Leptospira, the causative agents of leptospirosis, are responsible for over 1 million human cases annually and significantly impact domestic animals. Notably, bovine leptospirosis causes substantial financial losses due to abortion, stillbirths, and suboptimal reproductive performance. The advent of the CRISPR/Cas9 system has marked a turning point in genetic manipulation, with applications across multiple Leptospira species and serovars. However, incorporating controlled protein expression into existing genetic tools could further expand their utility. In this study, we developed and demonstrated the functionality of an IPTG-inducible heterologous protein expression system in Leptospira spp. This system was applied for regulated expression of dead Cas9 (dCas9) to generate knockdown mutants, and Cas9 to produce knockout mutants by inducing double-strand breaks (DSB) into desired targets. IPTG-induced dCas9 expression enabled validation of essential genes and non-coding RNAs in Leptospira. This approach, for the first time, distinguished between failed conjugation experiments and lethality caused by silencing essential genes, based on colony formation in the presence or absence of IPTG. Additionally, IPTG-controlled Cas9 expression combined with a constitutive non-homologous end-joining (NHEJ) system allowed for successful recovery of knockout mutants even without the inducer, due to minimal but undetectable Cas9 leakage. Unexpeectedly, knockout mutant recovery was favored by the absence of IPTG. These newly controlled protein expression systems will advance studies on the basic biology and virulence of Leptospira, as well as facilitate knockout mutant generation for improved veterinary vaccines.