Identification of proteins influencing CRISPR-associated transposases for enhanced genome editing

CRISPR-associated transposases (CASTs) hold tremendous potential for microbial genome editing because of their ability to integrate large DNA cargos in a programmable, site-specific manner. However, their widespread application has been hindered by poorly understood host factor requirements for transposition. To address this gap, we conducted the first genome-wide screen for host factors affecting Vibrio cholerae CAST ( Vch CAST) activity using an Escherichia coli RB-TnSeq library and identified 15 genes affecting Vch CAST transposition. Of these, seven factors were validated to improve Vch CAST activity, and two were inhibitory. Guided by the identification of homologous recombination effectors, RecD and RecA, we tested the λ-Red recombineering system in our Vch CAST editing vectors and increased editing efficiency by 55.2-fold in E. coli , 5.6-fold in Pseudomonas putida , and 10.8-fold in Klebsiella michiganensis while maintaining high target specificity and similar insertion arrangements. This study improves the understanding of factors affecting Vch CAST activity and enhances its efficiency as a bacterial genome editor.