Biology subjects

Swartjes, T.

Publications and source records attributed to Swartjes, T..

3 recordsLinked to original sources

bioRxiv2026

Transposon end recognition and excision mechanisms of type I-F CRISPR-associated transposases

CRISPR-associated transposons (CASTs) are Tn7-like elements that have co-opted RNA-guided CRISPR effectors for targeted DNA insertion. CASTs have been adapted as genome editing tools for programmable, site-specific integration. Among them, the type I-F system from Pseudoalteromonas (PseCAST) shows uniquely robust activity in human cells, yet its mechanistic basis remains poorly understood. Here, we present structural and biochemical analysis of the PseCAST transposase TnsAB. Biochemical reconstitution of transposon DNA excision defines key characteristics of the transposition mechanism. Cryogenic electron microscopy (cryo-EM) structures of PseTnsAB paired-end complexes reveal molecular determinants of transpososome assembly, transposon end recognition and cleavage. We validate these findings using biochemical and in vivo assays of structure-based transposase mutants, and provide mechanistic insights into the enhanced activity of a laboratory-evolved TnsAB variant. Together, our studies highlight molecular features underlying the efficiency of natural and engineered type I-F transposases and establish a mechanistic framework for their continued rational optimization.

biochemistry↗

bioRxiv2024

Towards sequential conjugation-assisted laboratory evolution (SCALE) of Cas nucleases

DNA endonucleases such as the CRISPR-associated enzymes Cas9 and Cas12a constitute powerful molecular tools with a wide range of applications. To facilitate optimization of these enzymes, we investigated a fully in vivo selection system for laboratory evolution based on plasmid conjugation. In the proposed method, DNA cleavage promotes plasmid transfer to other cells, allowing cleaving variants to spread through the bacterial culture via consecutive conjugations. We have investigated whether the conjugation system of the F plasmid of E. coli is suitable for laboratory evolution of Cas nucleases. Through a series of conjugation experiments, we found (i) that the F plasmid can spread through bacterial cultures, (ii) that the FinOP conjugation inhibition system is a potential nuclease target as it can prevent transfer of non-cleaving variants (counterselection), and (iii) that a trimmed version of the F plasmid allows for consecutive conjugations. Overall, we were able to show that in many regards, conjugation can indeed be used as means of in vivo laboratory evolution. We discuss the potential of the here presented concept for a yet to be developed laboratory evolution platform.

molecular biology↗

bioRxiv2022

Modulating CRISPR-Cas genome editing using guide-complementary DNA oligonucleotides

CRISPR-Cas has revolutionized genome editing and has a great potential for applications, such as correcting human genetic disorders. To increase the safety of genome editing applications, CRISPR-Cas may benefit from strict control over Cas enzyme activity. Previously, anti-CRISPR proteins and designed oligonucleotides have been proposed to modulate CRISPR-Cas activity. Here we report on the potential of guide-complementary DNA oligonucleotides as controlled inhibitors of Cas9 ribonucleoprotein complexes. First, we show that DNA oligonucleotides down-regulate Cas9 activity in human cells, reducing both on and off-target cleavage. We then used in vitro assays to better understand how inhibition is achieved and under which conditions. Two factors were found to be important for robust inhibition: the length of the complementary region, and the presence of a PAM-loop on the inhibitor. We conclude that DNA oligonucleotides can be used to effectively inhibit Cas9 activity both ex vivo and in vitro.

molecular biology↗