AM
A.R. Martins Costa
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1 records found
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Master thesis
(2020)
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Rodrigo Gonzalez Linares, A.R. Martins Costa, C. Almendros Romero, S.J.J. Brouns
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.
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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.