Researchers have developed a CRISPR-based approach that enables direct genome integration without generating double-strand breaks.
Programmable genome insertion is a key part of both gene therapy and research. Current methods to insert long DNA sequences are either inefficient or rely on double-strand break (DSB) repair. This requires programmable nucleases, such as CRISPR-Cas9, to induce repair pathways, such as non-homologous end joining (NHEJ) or homology directed repair (HDR). These approaches are limited as damage to the genome can result in undesirable outcomes, including insertions/deletions and translocations. In addition, NHEJ can generate off-target insertions at unintended breaks. HDR also has low efficiency in non-dividing cells and many cell types in vivo. It also requires long DNA templates that are labour-intensive to produce. As a result, researchers have been using newer technologies, such as base and prime editing, to avoid creating DSBs. However, these technologies cannot make all genome edits. These approaches are typically limited to modifications or insertions of short sequences.
Alternatively, natural transposable elements contain several families of integrases and transposes that could provide an efficient route for genome integration. However, these elements lack the programmability of CRISPR effector nucleases.
Combining CRISPR with integrases
In a recent study, published as preprint in biorxiv, researchers attempted to overcome the current limitations of gene integration approaches by combining the programmable nature of CRISPR with the efficient integration activity of integrases to create a precise DNA integration tool.
The tool known as PASTE, or Programmable Addition via Site-specific Targeting Elements, can achieve efficient and versatile gene integration at diverse loci. The tool specifically uses a CRISPR-Cas9 nickase fused with a reverse transcriptase and serine integrase to directly insert genetic sequences. PASTE can be delivered with a single dose of plasmids and is functional in both non-dividing and primary cells. It can also be easily retargeted to new genes. The team were able to integrate cargos of up to ~36kb in a single-delivery reaction. Here, they saw efficiencies of up to ~55% in a diversity of cell types. Overall, PASTE has editing efficiencies comparable to, or better than, those of HDR or NHEJ, and also has fewer detectable off-target effects.
To further improve PASTE, the team mined bacterial genomes and metagenomes to identify novel integrases which they applied as part of the system to enable high efficiency integration. They also showed that diverse templates, including those carried by AAV and adenovirus vectors, were compatible with PASTE. This allows for a drag-and-drop DNA integration of viruses and other DNA templates. This feature is particularly important for therapeutic purposes.
In summary, PASTE represents a new technology that expands on the capabilities of genome editing that enables efficient programmable gene integration at any targeted loci without DSBs. This approach provides a platform with broad applications in basic research, cell engineering and gene therapy.
Image credit: canva