Precise cut-and-paste DNA insertion using engineered type V-K CRISPR-associated transposases

CRISPR-associated transposases (CASTs) enable recombination-independent, multi-kilobase DNA insertions at RNA-programmed genomic locations. However, the utility of type V-K CASTs is hindered by high off-target integration and a transposition mechanism that results in a mixture of desired simple carg...

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Bibliographic Details
Published in:Nature biotechnology 2023-07, Vol.41 (7), p.968-979
Main Authors: Tou, Connor J., Orr, Benno, Kleinstiver, Benjamin P.
Format: Article
Language:English
Subjects:
631/1647
631/337/2569
631/45/607
631/61
Agriculture
Bioinformatics
Biomedical and Life Sciences
Biomedical Engineering/Biotechnology
Biomedicine
Biotechnology
Cargo
Clustered Regularly Interspaced Short Palindromic Repeats - genetics
CRISPR
CRISPR-Cas Systems - genetics
Deoxyribonucleic acid
DNA
DNA Transposable Elements
Endonucleases - genetics
Genomes
Genomics
Homing endonuclease
Humans
Insertion
Integration
Life Sciences
Nicking endonuclease
Plasmids
Purity
Recombination
Transposases - genetics
Transposases - metabolism
Transposition
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Description
Summary:CRISPR-associated transposases (CASTs) enable recombination-independent, multi-kilobase DNA insertions at RNA-programmed genomic locations. However, the utility of type V-K CASTs is hindered by high off-target integration and a transposition mechanism that results in a mixture of desired simple cargo insertions and undesired plasmid cointegrate products. Here we overcome both limitations by engineering new CASTs with improved integration product purity and genome-wide specificity. To do so, we engineered a nicking homing endonuclease fusion to TnsB (named HELIX) to restore the 5′ nicking capability needed for cargo excision on the DNA donor. HELIX enables cut-and-paste DNA insertion with up to 99.4% simple insertion product purity, while retaining robust integration efficiencies on genomic targets. HELIX has substantially higher on-target specificity than canonical CASTs, and we identify several novel factors that further regulate targeted and genome-wide integration. Finally, we extend HELIX to other type V-K orthologs and demonstrate the feasibility of HELIX-mediated integration in human cell contexts. Engineered CRISPR-associated transposases improve the purity and specificity of DNA insertions.
ISSN:1087-0156
1546-1696
DOI:10.1038/s41587-022-01574-x