Quantitative assay for TALEN activity at endogenous genomic loci

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Gene editing technologies like zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) enable targeted DNA double-strand breaks for gene disruption.
• Existing assays for evaluating nuclease activity on endogenous genomic loci are limited.

Purpose of the Study:

• To develop and validate a versatile modified lacZ assay for detecting frameshifts induced by genome editing nucleases.
• To provide a reliable method for assessing the activity of artificial nucleases at specific genomic locations.

Main Methods:

• Genomic DNA fragments from TALEN-treated or control embryos were amplified from target and off-target loci.
• Amplified fragments were inserted into the lacZα sequence for blue-white selection.
• Frameshift frequency was determined by colony counting, and insertions/deletions were identified via sequencing.

Main Results:

• The modified lacZ assay successfully detected frameshifts in nuclease target sites.
• The assay allowed for the estimation of frameshift frequency based on blue and white colony counts.
• Sequencing confirmed insertions and deletions, providing detailed information on gene editing outcomes.

Conclusions:

• The developed lacZ assay is a versatile tool for detecting nuclease-induced frameshifts.
• This method offers broad applicability for evaluating genome editing nucleases in various model organisms.
• The assay facilitates the assessment of gene disruption efficiency and accuracy in genome editing applications.