A CRISPR-associated transposase presents null cargo integration efficiency when targeting a transcriptionally highly active region

A CRISPR-associated transposase presents null cargo integration efficiency when targeting a transcriptionally highly active region

GONZALEZ LINARES, Rodrigo (TU Delft Applied Sciences; TU Delft BN/Bionanoscience; Kavli institute of nanoscience Delft)

Martins Costa, A.R. (mentor)
Almendros Romero, C. (mentor)
Brouns, S.J.J. (graduation committee)

Delft University of Technology

Applied Sciences | Nanobiology

2020-07-17

CRISPR-Cas effectors (e.g. Cas9) have been widely used to perform genetic knock-outs. Performing knock-ins however, remains challenging due to the inefficiency of the endogenous pathway cells use to integrate a donor genetic cargo into its genome (homology directed repair) when compared to other repair pathways like non-homologous end joining. CRISPR-associated transposases are complexes formed by a catalysis-deficient effector and a transposase. These complexes are able to sequester a transposon, localize a genomic target specified by a CRISPR RNA (crRNA), and integrate the transposon near the targeted site; thereby bypassing homology directed repair. In this study we aimed at developing a screening method using a CRISPR- associated transposase known as CAST, to detect integration events based on the disruption of lacZ. During the development, we found that CAST is unable to integrate a cargo in this highly active gene, most likely due to RNA polymerase-mediated dislodgment of the complex, and physical impediment for transposition proteins to reach the target DNA.

CRISPR-Cas
Transposase
Transposon

http://resolver.tudelft.nl/uuid:2ce74526-9fa0-4255-a763-6fe86416cec2

Student theses

master thesis

© 2020 Rodrigo GONZALEZ LINARES