AddTag, a two-step approach with supporting software package that facilitates CRISPR/Cas-mediated precision genome...

Area of Science:

• Molecular Biology
• Genetics
• Bioengineering

Background:

• CRISPR/Cas genome editing offers powerful genetic engineering capabilities but faces limitations due to targeting constraints.
• Precision editing of small genomic features is challenging because DNA sequence length affects the probability of overlapping unique target sites.

Purpose of the Study:

• To introduce a novel two-step genome editing strategy that overcomes CRISPR/Cas targeting limitations.
• To enable precision genome editing of small genomic elements at virtually any locus.

Main Methods:

• A two-step genome editing strategy involving an "AddTag" sequence replacement.
• The AddTag sequence is first inserted at the locus of interest and then replaced with a desired engineered sequence.
• Demonstration in Candida albicans by editing transcription factor binding sites.

Main Results:

• The AddTag approach effectively eliminates CRISPR/Cas targeting constraints.
• Enabled precise editing of single base-pair genomic elements.
• Successfully edited transcription factor binding sites in Candida albicans that were previously inaccessible.
• Demonstrated utility for combinatorial genome editing and gene complementation analysis.
• Developed a software package to automate AddTag editing design.

Conclusions:

• The AddTag strategy significantly expands the scope and precision of CRISPR/Cas genome editing.
• This method facilitates the editing of previously intractable genomic loci and small genetic elements.
• The approach has broad applicability across organisms supporting CRISPR/Cas and aids in complex genetic analyses.