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

Abstract

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 <i>Vibrio cholerae</i> CAST (<i>Vch</i>CAST) activity using an <i>Escherichia coli</i> RB-TnSeq library and identified 15 genes affecting <i>Vch</i>CAST transposition. Of these, seven factors were validated to improve <i>Vch</i>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 <i>Vch</i>CAST editing vectors and increased editing efficiency by 55.2-fold in <i>E. coli</i>, 5.6-fold in <i>Pseudomonas putida</i>, and 10.8-fold in <i>Klebsiella michiganensis</i> while maintaining high target specificity and similar insertion arrangements. This study improves the understanding of factors affecting <i>Vch</i>CAST activity and enhances its efficiency as a bacterial genome editor.