In vivo delivery of functional Cas:DNA nucleoprotein complexes into recipient bacteria through a Type IV Secretion System

Abstract

AbstractCRISPR-associated (Cas) endonucleases and their derivatives are widespread tools for the targeted genetic modification of both prokaryotic and eukaryotic genomes. A critical step of all CRISPR-Cas technologies is the delivery of the Cas endonuclease to the target cell. Here, we investigate the possibility of using bacterial conjugation to translocate Cas proteins into recipient bacteria. Conjugative relaxases are translocated through a Type IV Secretion System (T4SS) into the recipient cell, covalently attached to the transferred DNA strand. We fused relaxase R388-TrwC with the class 2 Cas endonuclease Cas12a and confirmed that it can be transported through a T4SS. The fusion protein maintained its activity upon translocation by conjugation into the recipient cell, as evidenced by the induction of the SOS signal resulting from the cuts produced by the endonuclease in the recipient cell, and the detection of mutations at the target position. We further show how a template DNA provided on the transferred DNA can be used to introduce specific mutations. The gRNA can also be encoded by the transferred DNA, enabling its production in the recipient cells where it can form a complex with the Cas nuclease transferred as a protein. This self-contained setup enables to target wild type bacterial cells. Finally, we extended this strategy to the delivery of relaxases fused to base editors. Using both TrwC and MobA relaxases as drivers, we achieved precise editing of transconjugants. Thus, conjugation provides anin vivodelivery system for Cas-derived editing tools, bypassing the need to deliver and express acasgene in the target cells.Significance StatementWe have developed a novel approach for introducing CRISPR-Cas genetic tools into bacteria. During bacterial conjugation, the relaxase protein is transferred through the secretion system covalently attached to the transferred DNA. By fusing the Cas protein with the conjugative relaxase, we have observed functional Cas activity in the recipient cells, eliminating the need for nuclease expression in these cells. The covalently attached DNA molecule can supply gRNA and donor DNA, enabling seamless genetic modifications through recombination. We have also successfully translocated fusions of relaxases to base editors which are active in recipient cells. This method can be applied to any potential recipient cells, making it particularly interesting for wild type bacterial strains that lack available genetic tools. Furthermore, this method has the potential to be extended to eukaryotic cells.