Homology-independent targeted insertion (HITI) is a CRISPR/Cas9-based strategy that enables gene insertion in non-dividing cells in vivo and in vitro. Site-specific transgene integration can be achieved by the homology-directed repair (HDR) pathway such as short-fragment homologous recombination (SFHR). This process is inefficient and the process is not active in non-dividing cells. The other major double strand break (DSB) repair pathway, non-homologous end joining (NHEJ), is active in both proliferating and non-dividing cells and is generally more efficient than HDR in mammals. Whereas HDR can replace the target sequence, NHEJ can add an ectopic DNA sequence at the target locus instead of replacing the original DNA sequence. However NHEJ is recognized as error-prone.
HITI uses an NHEJ-based homology-independent strategy. Research lead by Juan Carlos Izpisua Belmonte at the Salk Institute for Biological Studies in La Jolla, California suggests that error-free repair is dominant in their CRISPR/Cas9-based HITI method and that the method is more efficient than HDR. Guide RNAs (gRNAs) were used to specify the direction of the insertion. Classical HDR-based CRISPR/Cas9 genome-editing involves transfecting cells with Cas9, gRNA and donor DNA containing homologous arms matching the genomic locus of interest. For HITI, donor plasmids lack homology arms and DSB does not occur through the HDR pathway. The donor DNA includes Cas9 cleavage site(s) flanking the donor sequence resulting in Cas9 cleavage at both the donor plasmid and the genomic target sequence. Both target and donor have blunt ends and the linearized donor DNA plasmid is used by the NHEJ pathway resulting integration into the genomic DSB site. When incorporated in the correct orientation, the Cas9 target sequence is disrupted preventing further Cas9 cutting.
The HITI strategy was demonstrated in neurons and was the first demonstration of gene knock-in in a non-dividing cell type. The HITI method has been used to successfully treat a loss-of-function mutation in a rat model for retinitis pigmentosa. In a review article on HITI in 2018, the authors note low efficiency and potential for off-target integration as a barrier to overcome before HITI will be successful in clinical applications.
In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration
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- CRISPR/Cas ToolsClustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated (Cas) proteins perform adaptive immune functions in prokaryotic organisms defending against foreign nucleic acids such as viruses. CRISPR/Cas tools have been adapted for use in genome editing and other DNA and RNA targeting applications.
- Cluster: BiotechnologyA cluster of topics related to biotechnology.
- CRISPR-Cas9CRISPR-Cas9 is a genome editing system. CRISPR systems provides immunity to bacteria and archaea from viruses and has been adapted for use as a genome editing tool capable of knocking out genes and rewriting genetic sequences in animal, plant and fungi. CRISPR-Cas9 is being adapted to other applications outside genome editing.
- Gene therapyGene Therapy uses genes to treat or prevent disease. Types of gene therapy include replacing a mutated gene with a healthy copy, inactivating or knocking out a mutated gene or introducing a new gene to fight disease.