Efficient and Scalable Precision Genome Editing in through Conditional Recombineering and CRISPR/Cas9-Mediated Counterselection.

is an important human pathogen, but studies of the organism have suffered from the lack of a robust tool set for its genetic and genomic manipulation. Here we report the development of a system for the facile and high-throughput genomic engineering of using single-stranded DNA (ssDNA) oligonucleotide recombineering coupled with clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated counterselection. We identify recombinase , derived from , as being capable of integrating single-stranded DNA oligonucleotides into the genome. We found that can readily mediate recombineering across multiple characterized strains (3 of 3 tested) and primary clinical isolates (6 of 6 tested), typically yielding thousands of recombinants per transformation. Surprisingly, we also found that some strains are naturally recombinogenic at measurable frequencies when oligonucleotides are introduced by electroporation, even without exogenous recombinase expression. We construct a temperature-sensitive, two-vector system which enables conditional recombineering and CRISPR/Cas9-mediated counterselection in without permanently introducing exogenous genetic material or unintended genetic lesions. We demonstrate the ability of this system to efficiently and precisely engineer point mutations and large single-gene deletions in the genome and to yield highly enriched populations of engineered recombinants even in the absence of an externally selectable phenotype. By virtue of utilizing inexpensive, commercially synthesized synthetic DNA oligonucleotides as substrates for recombineering and counterselection, this system provides a scalable, versatile, precise, inexpensive, and generally useful tool for producing isogenic strains in which will enable the high-throughput functional assessment of genome variation and gene function across multiple strain backgrounds. Engineering genetic changes in bacteria is critical to understanding the function of particular genes or mutations but is currently a laborious and technically challenging process to perform for the important human pathogen In an effort to develop methods which are rapid, easy, scalable, versatile, and inexpensive, here we describe a system for incorporating synthetic, mutagenic DNA molecules into the genome and for eliminating cells that lack the engineered mutation. This method allows efficient, precise, and high-throughput genetic engineering of strains and will facilitate studies seeking to address a variety of issues about the function of particular genes and specific mutations.