CRISPR-Mediated Cassette Exchange (CriMCE): A Method to Introduce and Isolate Precise Marker-Less Edits

Ioanna Morianou; Andrea Crisanti; Tony Nolan; Andrew M. Hammond

doi:10.1089/crispr.2022.0026

CRISPR-Mediated Cassette Exchange (CriMCE): A Method to Introduce and Isolate Precise Marker-Less Edits

Ioanna Morianou, Andrea Crisanti, Tony Nolan, Andrew M. Hammond

Vector Biology

Research output: Contribution to journal › Article › peer-review

3 Citations (Scopus)

Abstract

The introduction of small unmarked edits to the genome of insects is essential to study the molecular underpinnings of important biological traits, such as resistance to insecticides and genetic control strategies. Advances in CRISPR genome engineering have made this possible, but prohibitively laborious for most laboratories due to low rates of editing and the lack of a selectable marker. To facilitate the generation and isolation of precise marker-less edits we have developed a two-step method based on CRISPR-mediated cassette exchange (CriMCE) of a marked placeholder for a variant of interest. This strategy can be used to introduce a wider range of potential edits compared with previous approaches while consolidating the workflow. We present proof-of-principle that CriMCE is a powerful tool by engineering three single nucleotide polymorphism variants into the genome of Anopheles gambiae, with 5–41 × higher rates of editing than homology-directed repair or prime editing.

Original language	English
Pages (from-to)	868-876
Journal	CRISPR Journal
Volume	5
Issue number	6
Early online date	15 Nov 2022
DOIs	https://doi.org/10.1089/crispr.2022.0026
Publication status	Published - 12 Dec 2022

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abstract = "The introduction of small unmarked edits to the genome of insects is essential to study the molecular underpinnings of important biological traits, such as resistance to insecticides and genetic control strategies. Advances in CRISPR genome engineering have made this possible, but prohibitively laborious for most laboratories due to low rates of editing and the lack of a selectable marker. To facilitate the generation and isolation of precise marker-less edits we have developed a two-step method based on CRISPR-mediated cassette exchange (CriMCE) of a marked placeholder for a variant of interest. This strategy can be used to introduce a wider range of potential edits compared with previous approaches while consolidating the workflow. We present proof-of-principle that CriMCE is a powerful tool by engineering three single nucleotide polymorphism variants into the genome of Anopheles gambiae, with 5–41 × higher rates of editing than homology-directed repair or prime editing.",

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AU - Hammond, Andrew M.

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CRISPR-Mediated Cassette Exchange (CriMCE): A Method to Introduce and Isolate Precise Marker-Less Edits

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