A CRISPR-associated transposase presents null cargo integration efficiency when targeting a transcriptionally highly active region
R. GONZALEZ LINARES (TU Delft - Applied Sciences)
Ana Rita Martins Costa – Mentor (TU Delft - BN/Stan Brouns Lab)
C. Almendros Romero – Mentor (TU Delft - BN/Stan Brouns Lab)
Stan J. J. Brouns – Graduation committee member (TU Delft - BN/Stan Brouns Lab)
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Abstract
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.