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Genome Editing in Mammalian Cell Lines using CRISPR-Cas

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Precise kilobase-scale genomic insertions in mammalian cells using PASTE.

Christopher W Fell^1,2,3,4, Cian Schmitt-Ulms^1,2,3,4,5, Dario V Tagliaferri^1,2,3,4

¹Harvard Medical School, Harvard University, Boston, MA, USA.

Nature Protocols

|December 15, 2024

Summary

Programmable Addition via Site-Specific Targeting Elements (PASTE) enables precise, efficient, and large DNA insertions into mammalian genomes. This technology offers improved editing, purity, and programmability for gene integration applications.

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Area of Science:

Molecular Biology
Genetics
Biotechnology

Background:

Programmable gene integration is crucial for research and therapeutics.
Existing methods struggle with large DNA insertions into mammalian genomes.

Purpose of the Study:

To introduce and detail the Programmable Addition via Site-Specific Targeting Elements (PASTE) technology.
To provide a comprehensive protocol for utilizing PASTE for precise gene integration.

Main Methods:

PASTE combines site-specific integrases with prime editing for targeted DNA insertion.
Protocols detail EGFP integration at human NOLC1 and ACTB loci.
Readout methods include next-generation sequencing and droplet digital PCR.

Main Results:

PASTE achieves precise insertion of large DNA cargoes (≥36 kb) with up to 60% efficiency.
Demonstrated improved editing efficiency, purity, and programmability over previous methods.
Successful applications include in-frame protein tagging and multiplexed insertions.

Conclusions:

PASTE is a versatile and efficient tool for large DNA sequence integration in mammalian genomes.
The protocol facilitates custom PASTE experiments for diverse genomic loci and payloads.
PASTE enables rapid (2-week) and precise gene insertion for research and therapeutic development.