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Targeted DNA integration in human cells without double-strand breaks using CRISPR RNA-guided transposases

Traditional genome-editing reagents such as CRISPR-Cas9 achieve targeted DNA modification by introducing double-strand breaks (DSBs), thereby stimulating localized DNA repair by endogenous cellular repair factors. While highly effective at generating heterogenous knockout mutations, this approach su...

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Autores principales: Lampe, George D., King, Rebeca T., Halpin-Healy, Tyler S., Klompe, Sanne E., Hogan, Marcus I., Vo, Phuc Leo H., Tang, Stephen, Chavez, Alejandro, Sternberg, Samuel H.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Cold Spring Harbor Laboratory 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10055298/
https://www.ncbi.nlm.nih.gov/pubmed/36993517
http://dx.doi.org/10.1101/2023.03.17.533036
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author Lampe, George D.
King, Rebeca T.
Halpin-Healy, Tyler S.
Klompe, Sanne E.
Hogan, Marcus I.
Vo, Phuc Leo H.
Tang, Stephen
Chavez, Alejandro
Sternberg, Samuel H.
author_facet Lampe, George D.
King, Rebeca T.
Halpin-Healy, Tyler S.
Klompe, Sanne E.
Hogan, Marcus I.
Vo, Phuc Leo H.
Tang, Stephen
Chavez, Alejandro
Sternberg, Samuel H.
author_sort Lampe, George D.
collection PubMed
description Traditional genome-editing reagents such as CRISPR-Cas9 achieve targeted DNA modification by introducing double-strand breaks (DSBs), thereby stimulating localized DNA repair by endogenous cellular repair factors. While highly effective at generating heterogenous knockout mutations, this approach suffers from undesirable byproducts and an inability to control product purity. Here we develop a system in human cells for programmable, DSB-free DNA integration using Type I CRISPR-associated transposons (CASTs). To adapt our previously described CAST systems, we optimized DNA targeting by the QCascade complex through a comprehensive assessment of protein design, and we developed potent transcriptional activators by exploiting the multi-valent recruitment of the AAA+ ATPase, TnsC, to genomic sites targeted by QCascade. After initial detection of plasmid-based transposition, we screened 15 homologous CAST systems from a wide range of bacterial hosts, identified a CAST homolog from Pseudoalteromonas that exhibited improved activity, and increased integration efficiencies through parameter optimization. We further discovered that bacterial ClpX enhances genomic integration by multiple orders of magnitude, and we propose that this critical accessory factor functions to drive active disassembly of the post-transposition CAST complex, akin to its demonstrated role in Mu transposition. Our work highlights the ability to functionally reconstitute complex, multi-component machineries in human cells, and establishes a strong foundation to realize the full potential of CRISPR-associated transposons for human genome engineering.
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spelling pubmed-100552982023-03-30 Targeted DNA integration in human cells without double-strand breaks using CRISPR RNA-guided transposases Lampe, George D. King, Rebeca T. Halpin-Healy, Tyler S. Klompe, Sanne E. Hogan, Marcus I. Vo, Phuc Leo H. Tang, Stephen Chavez, Alejandro Sternberg, Samuel H. bioRxiv Article Traditional genome-editing reagents such as CRISPR-Cas9 achieve targeted DNA modification by introducing double-strand breaks (DSBs), thereby stimulating localized DNA repair by endogenous cellular repair factors. While highly effective at generating heterogenous knockout mutations, this approach suffers from undesirable byproducts and an inability to control product purity. Here we develop a system in human cells for programmable, DSB-free DNA integration using Type I CRISPR-associated transposons (CASTs). To adapt our previously described CAST systems, we optimized DNA targeting by the QCascade complex through a comprehensive assessment of protein design, and we developed potent transcriptional activators by exploiting the multi-valent recruitment of the AAA+ ATPase, TnsC, to genomic sites targeted by QCascade. After initial detection of plasmid-based transposition, we screened 15 homologous CAST systems from a wide range of bacterial hosts, identified a CAST homolog from Pseudoalteromonas that exhibited improved activity, and increased integration efficiencies through parameter optimization. We further discovered that bacterial ClpX enhances genomic integration by multiple orders of magnitude, and we propose that this critical accessory factor functions to drive active disassembly of the post-transposition CAST complex, akin to its demonstrated role in Mu transposition. Our work highlights the ability to functionally reconstitute complex, multi-component machineries in human cells, and establishes a strong foundation to realize the full potential of CRISPR-associated transposons for human genome engineering. Cold Spring Harbor Laboratory 2023-03-18 /pmc/articles/PMC10055298/ /pubmed/36993517 http://dx.doi.org/10.1101/2023.03.17.533036 Text en https://creativecommons.org/licenses/by-nc-nd/4.0/This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (https://creativecommons.org/licenses/by-nc-nd/4.0/) , which allows reusers to copy and distribute the material in any medium or format in unadapted form only, for noncommercial purposes only, and only so long as attribution is given to the creator.
spellingShingle Article
Lampe, George D.
King, Rebeca T.
Halpin-Healy, Tyler S.
Klompe, Sanne E.
Hogan, Marcus I.
Vo, Phuc Leo H.
Tang, Stephen
Chavez, Alejandro
Sternberg, Samuel H.
Targeted DNA integration in human cells without double-strand breaks using CRISPR RNA-guided transposases
title Targeted DNA integration in human cells without double-strand breaks using CRISPR RNA-guided transposases
title_full Targeted DNA integration in human cells without double-strand breaks using CRISPR RNA-guided transposases
title_fullStr Targeted DNA integration in human cells without double-strand breaks using CRISPR RNA-guided transposases
title_full_unstemmed Targeted DNA integration in human cells without double-strand breaks using CRISPR RNA-guided transposases
title_short Targeted DNA integration in human cells without double-strand breaks using CRISPR RNA-guided transposases
title_sort targeted dna integration in human cells without double-strand breaks using crispr rna-guided transposases
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10055298/
https://www.ncbi.nlm.nih.gov/pubmed/36993517
http://dx.doi.org/10.1101/2023.03.17.533036
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