AddTag

AddTag enables two-step precision genome editing to permit precise modification of genomic loci, including small DNA sequences lacking unique CRISPR/Cas target sites.

Key Features:

• Two-Step Editing Strategy: Replaces the target genomic locus with an AddTag sequence and subsequently substitutes the AddTag with any engineered DNA sequence using CRISPR/Cas.
• Elimination of Targeting Constraints: Circumvents the requirement for a unique CRISPR/Cas target site, enabling precision editing of genomic elements as small as a single base-pair.
• Versatility Across Organisms: Applicable to any organism that supports CRISPR/Cas-mediated genome editing.
• Software Package: Includes a supporting software package that automates the design of the AddTag editing process.

Scientific Applications:

• Precision Editing of Small Genomic Features: Enables editing of small elements such as transcription factor binding sites that are challenging to target with conventional CRISPR/Cas methods.
• Combinatorial Genome Editing: Facilitates multiple genetic modifications performed simultaneously or sequentially within the same organism.
• Gene Complementation Analysis: Allows precise insertional modifications to support complementation studies for elucidating gene function and interactions.
• Targeted Research Example: Has been applied to edit transcription factor binding sites in Candida albicans that were previously uneditable using conventional gene-editing techniques.

Methodology:

Designing an "AddTag" sequence tailored to the target locus; using CRISPR/Cas to replace the original genomic sequence with this AddTag; and performing a second round of CRISPR/Cas editing to substitute the AddTag with the desired genetic modification.