A method and system for constructing high-quality whole-genome knockout collections for functional genomics in microorganisms

Background

The field of functional genomics seeks to understand the roles of genes within an organism by systematically disrupting gene function and observing the resulting phenotypes. This approach is critical for deciphering gene function, identifying drug targets, and advancing synthetic biology. Traditionally, curated collections of single-gene knockout mutants have been invaluable for model organisms, enabling researchers to link gene sequence to function on a genome-wide scale. However, despite the rapid growth in genome sequencing, a significant portion of genes—often 30-40%—remain uncharacterized, particularly in non-model or newly sequenced microorganisms. The ability to generate comprehensive knockout collections for a wide variety of organisms is essential for unlocking the functional potential of these genomes and driving innovation in biotechnology and medicine.

Current methods for constructing whole-genome knockout collections face substantial limitations. Targeted gene deletion techniques, such as those used to create the Yeast Deletion Collection or the Keio Collection for E. coli, are labor-intensive, expensive, and require years to complete, restricting their use to a handful of well-studied organisms. Alternative approaches using transposon mutagenesis demand massive oversampling—often tens of thousands of mutants for a single genome—and involve complex, error-prone annotation processes. These methods are not only cost-prohibitive but also impractical for most laboratories, especially when working with non-model organisms that lack established genetic tools. As a result, the creation of high-quality, non-redundant knockout collections remains out of reach for many researchers, limiting the pace and breadth of functional genomics research.

Technology

This technology is a comprehensive platform for rapidly constructing whole-genome knockout collections in microorganisms by integrating transposon mutagenesis with a four-dimensional combinatorial pooling strategy and advanced Bayesian inference algorithms. The process begins by generating a large library of mutant strains through transposon insertion, followed by organizing these mutants into pools based on their plate and well coordinates. This pooling scheme enables unique identification of each mutant using next-generation sequencing. Sequencing data is then analyzed with Bayesian inference tools that accurately map each genomic insertion to its physical location, even in the presence of cross-contamination or duplicate samples. Selection algorithms identify the optimal mutant for each gene—favoring those with disruptions closest to the translation start site—to create a condensed, non-redundant collection. The final step involves orthogonal validation, where the condensed library is re-sequenced to confirm the accuracy and purity of the gene disruptions.

What differentiates this technology is its dramatic increase in speed, scalability, and affordability compared to traditional methods for constructing knockout collections. Unlike labor-intensive and costly targeted gene deletion techniques, this approach leverages high-throughput combinatorial pooling and automated data analysis to process tens of thousands of mutants in a fraction of the time and at a fraction of the cost. The use of Bayesian inference ensures robust mapping of mutants, even in complex or noisy datasets, while the orthogonal validation step guarantees high-quality, contamination-free collections. This platform is broadly applicable to a wide range of microorganisms, including non-model species, and is supported by open-source software and detailed protocols, making it accessible to most laboratories. Its ability to generate highly complete, non-redundant knockout collections democratizes functional genomics, enabling rapid gene function discovery, drug target identification, and synthetic biology applications.

Benefits

• Rapid and cost-effective construction of whole-genome knockout collections, reducing time and expense by 10-1000 times compared to traditional methods.
• High-throughput and scalable approach capable of processing tens of thousands of mutants daily using a simple combinatorial pooling strategy.
• Accurate identification and mapping of mutants through Bayesian inference algorithms that handle cross-contamination and duplicate samples.
• Generation of highly complete, non-redundant, and contamination-free knockout libraries suitable for diverse microorganisms.
• Orthogonal validation via re-sequencing ensures high quality and purity of the final mutant collections.
• Enables functional genomics, gene function discovery, drug target identification, and synthetic biology applications, especially in non-model organisms.
• Accessible protocol and software tools facilitate adoption by many laboratories without requiring specialized equipment or excessive costs.

Applications

• Functional genomics in non-model microbes
• High-throughput drug target discovery
• Synthetic biology parts screening
• Comparative genomics across species
• Industrial strain optimization

Additional info

This methodology generates whole-genome knockout collections by integrating transposon mutagenesis with four-dimensional combinatorial pooling and Bayesian inference. Mutant libraries are organized into coordinate-based pools for next-generation sequencing. Bayesian algorithms map genomic insertion sites to physical locations, resolving contamination. Selection algorithms prioritize optimal mutants to assemble condensed, non-redundant collections. Orthogonal validation through re-sequencing ensures high accuracy and purity, facilitating rapid, low-cost functional genomics in microorganisms.