Dual CRISPR Strategy Avoiding Lethal DNA Cleavage Opens Path for Precise Leptospira Editing
Background
Bacteria of the genus Leptospira are pathogens that cause leptospirosis in humans and livestock. They colonize the kidneys of various animals and are excreted in urine, contaminating the environment, but the genes responsible for pathogenicity and host adaptation remain poorly characterized. This is largely due to the difficulty in generating mutant strains to directly confirm gene function.
In conventional bacterial genome editing, Cas9 cuts both strands of the target DNA, and the cell's repair process induces gene disruption. However, most Leptospira cannot properly repair double-strand breaks (DSBs). Even when Cas9 accurately cuts the target, edited cells often die, making it difficult to recover knockout strains. CRISPR interference using catalytically inactive Cas9 can suppress gene expression, but it does not permanently alter the DNA sequence itself.
To overcome this limitation, the research team developed a dual CRISPR system combining CRISPR/Cas9-NHEJ and CRISPR prime editing (PE), which operate on different principles. The former provides DSB repair capability externally, while the latter avoids lethal DSBs altogether.
Key Findings
In the CRISPR/Cas9-NHEJ system, Cas9 and guide RNA are coexpressed with DNA repair proteins LigD and Ku from Mycobacterium smegmatis. Ku captures the cut DNA ends, and LigD connects them, thereby supplementing the deficient NHEJ function in Leptospira. This repair process is error-prone, resulting in insertions or deletions (indels) at the target site, which can disrupt the gene reading frame and generate knockout mutants.
PE is a more precise approach that reduces the risks of cutting and repairing DNA. The researchers fused a Cas9 nickase with a reverse transcriptase. A prime editing guide RNA directs the system to the target, and the reverse transcriptase records the desired sequence into new DNA. Since only one DNA strand is cut, this method preserves cell viability in DSB-sensitive Leptospira.
The two systems serve distinct roles. Cas9-NHEJ is well-suited for rapidly generating diverse indels through error-prone repair. In contrast, PE is ideal for experiments requiring precise, pre-designed outcomes, such as single-base substitutions. Unlike previous studies that relied on random mutagenesis or transient gene suppression, this new toolkit allows researchers to selectively choose between gene disruption and precise correction based on their experimental goals.
Significance and Outlook
The new editing systems provide a robust foundation for causally validating pathogenic factors, metabolic pathways, environmental survival, and host colonization genes in Leptospira. Comparing strains with complete gene deletions and those with specific base changes allows the effects of protein loss and individual amino acid alterations to be analyzed separately. The systems also offer potential for studying functional differences among various species and serovars.
In vaccine development, the systems could be used to create attenuated live vaccines by removing pathogenic genes or to engineer strains with modulated antigen production. However, the current achievements focus on expanding the editing principles and tools. Factors such as editing efficiency per target, off-target mutations, and strain-specific plasmid delivery and expression require further validation. Indels generated by NHEJ are variable, necessitating individual sequence confirmation for candidate strains, and PE performance can also vary depending on the target sequence and guide design. To assess the practical applicability of these systems, the genetic stability and pathogenicity changes of edited strains must be confirmed in animal infection models.
Genetic manipulation of Leptospira spp. has progressed significantly in recent years. Like most prokaryotes, leptospires are unable to survive double-strand breaks (DSBs) induced by the Cas9 endonuclease, prompting the development of alternative strategies for gene knockout. We have established two systems for targeted mutagenesis of Leptospira spp.: CRISPR/Cas9-NHEJ and CRISPR-Prime Editing (PE). The CRISPR/Cas9-NHEJ approach involves coexpression of the CRISPR/Cas9 machinery alongside the DNA repair proteins LigD and Ku from Mycobacterium smegmatis, facilitating error-prone repair of DSBs that results in indel mutations. In contrast, CRISPR-PE is a DSB-free strategy that utilizes a Cas9-nickase fused to a reverse transcriptase that facilitates precise single-nucleotide edits in the genome. This expanded toolbox has placed Leptospira spp. at the forefront of bacterial genetic manipulation.
In the laboratory, researchers can first use Cas9-NHEJ to delete suspected pathogenic genes and screen for infection phenotypes, then use PE to modify specific bases or amino acids in active regions to narrow down functional mechanisms. For example, by administering edited strains to hamster infection models and comparing their ability to induce acute disease and colonize the kidneys, the roles of genes at different stages of infection can be distinguished.
In industrial applications, designing vaccine strains by removing toxin-related genes without markers is a promising application. However, factors such as the stability of production strains during serial cultivation, off-target mutations, and environmental release risks must be evaluated. Since these pathogens are being precisely edited, biosafety management and regulatory standards must also be established in parallel.