Precise and Scarless Insertion of Transposable Elements by Cas9-Mediated Genome Engineering

Area of Science:

• Genetics
• Molecular Biology
• Genomics

Background:

• Transposable elements (TEs) significantly impact gene expression and can cause disease by altering regulatory elements.
• Understanding TE mechanisms requires precise genetic manipulation tools.
• Current methods for TE insertion often leave genetic scars or reporter genes.

Purpose of the Study:

• To establish a scarless, markerless method for precisely inserting transposable elements into the human genome.
• To enable the study of TE functions and regulatory roles in cultured human cells.
• To provide a versatile platform for knocking in large DNA sequences without genetic markers.

Main Methods:

• A two-step CRISPR-Cas9 gene editing approach was employed.
• Initially, a double-selection cassette was inserted at the target locus.
• Subsequently, the cassette was exchanged for a markerless transposable element sequence.

Main Results:

• Precise insertion of transposable elements into cultured human cells was achieved.
• The method successfully generated markerless and scarless insertions.
• The approach demonstrated versatility for knocking in large DNA inserts.

Conclusions:

• This CRISPR-Cas9-mediated strategy offers a robust tool for precise, markerless transposable element integration.
• The method facilitates the investigation of TE functions and their impact on gene regulation.
• This technique has broader applications for inserting large DNA fragments without residual genetic markers.