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Homology-independent targeted insertion (HITI)

Homology-independent targeted insertion (HITI)

HITI is a CRISPR/Cas9-based strategy that enables gene insertion in non-dividing cells in vivo and in vitro and has applications in gene therapy.

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Will Suter
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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 Cas9Cas9, 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. 

Will Suter
Will Suter approved a suggestion from Golden's AI on 20 Jun 2019 5:45 pm
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HITI uses an NHEJ-based homology-independent strategy. Research lead by Juan Carlos Izpisua Belmonte at the Salk Institute for Biological Studies in La JollaLa 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. 

Will Suter
Will Suter approved a suggestion from Golden's AI on 20 Jun 2019 5:44 pm
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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, CaliforniaCalifornia 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. 

Will Suter
Will Suter approved a suggestion from Golden's AI on 20 Jun 2019 5:42 pm
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HITI uses an NHEJ-based homology-independent strategy. Research lead by Juan Carlos Izpisua BelmonteJuan 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. 

Will Suter
Will Suter approved a suggestion from Golden's AI on 20 Jun 2019 5:42 pm
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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 DNADNA sequence at the target locus instead of replacing the original DNA sequence. However NHEJ is recognized as error-prone. 

Meredith Hanel"Made a concise title for this technology. Reworded text to not include words such as we, our and new. Previous text was copied directly but I have reworded and elaborated on the technology. Review article in timeline moved to further reading."
Meredith Hanel edited on 20 Jun 2019 2:21 pm
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In vivo genome editing via the HITI method as a tool for gene therapy Homology-independent targeted insertion (HITI)

Using genome-editing technologies to correct specific mutations represents a potentially transformative new approach for treating genetic disorders. Despite rapid advances in the field of genome editing, it is still unclear whether the long-standing goal of in vivo targeted transgene integration is feasible. This is primarily because

HITI is a CRISPR/Cas9-based strategy that enables gene insertion in non-dividing cells in vivo and in vitro and has applications in gene therapy.

Article

Using genome-editing technologies to correct specific mutations represents a potentially transformative new approach for treating genetic disorders. Despite rapid advances in the field of genome editing, it is still unclear whether the long-standing goal of in vivo targeted transgene integration is feasible. This is primarily because current tools are inefficient. In particular, current technologies are incapable of targeted gene knock-in in non-dividing cells, the major building blocks of adult tissues. This poses a significant barrier for developing therapeutic strategies to treat a broad range of devastating genetic disorders. Recently, our group has developed a unique CRISPR/Cas9-based strategy, termed homology-independent targeted insertion (HITI), which enables targeted gene insertion in non-dividing cells, both in vitro and in vivo. This review will summarize current progress in developing this technology, and discuss the potential impact of HITI-based gene-correction therapies.

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. 

...

Li J, Zhang B, Ren Y, Gu S, Xiang Y, Du J. Intron targeting-mediated and endogenous gene integrity-maintaining knockin in zebrafish using the CRISPR/Cas9 system. Cell Res. 2015;25:634-7.

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. 

...

Suzuki K, Tsunekawa Y, Hernandez-Benitez R, Wu J, Zhu J, Kim EJ, et al. In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration. Nature. 2016;540:144-9.

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. 

...

Yao X, Wang X, Liu J, Hu X, Shi L, Shen X, et al. CRISPR/Cas9 - mediated precise targeted integration in vivo using a double cut donor with short homology arms. EBioMedicine. 2017;20:19-26.

...

Yao X, Wang X, Hu X, Liu Z, Liu J, Zhou H, et al. Homology-mediated end joining-based targeted integration using CRISPR/Cas9. Cell Res. 2017;27:801-14.

...

Luo J, Baranov P, Patel S, Ouyang H, Quach J, Wu F, et al. Human retinal progenitor cell transplantation preserves vision. J Biol Chem. 2014;289:6362-71.

















Further reading

Title
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Type
Date

In vivo genome editing via the HITI method as a tool for gene therapy. - PubMed - NCBI

Suzuki K and Izpisua Belmonte JC

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Date invented
December 1, 2016

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November 13, 2017

In vivo genome editing via the HITI method as a tool for gene therapy

November 16, 2016

In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration

March 7, 2014

Human Retinal Progenitor Cell Transplantation Preserves Vision

Edits on 3 May 2019
Martin Bosnev
Martin Bosnev edited on 3 May 2019 12:27 pm
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Topic thumbnail

Homology-independent targeted insertion (HITI)

Using genome-editing technologies to correct specific mutations represents a potentially transformative new approach for treating genetic disorders. Despite rapid advances in the field of genome editing, it is still unclear whether the long-standing goal of in vivo targeted transgene integration is feasible. This is primarily because

Article

Using genome-editing technologies to correct specific mutations represents a potentially transformative new approach for treating genetic disorders. Despite rapid advances in the field of genome editing, it is still unclear whether the long-standing goal of in vivo targeted transgene integration is feasible. This is primarily because current tools are inefficient. In particular, current technologies are incapable of targeted gene knock-in in non-dividing cells, the major building blocks of adult tissues. This poses a significant barrier for developing therapeutic strategies to treat a broad range of devastating genetic disorders. Recently, our group has developed a unique CRISPR/Cas9-based strategy, termed homology-independent targeted insertion (HITI), which enables targeted gene insertion in non-dividing cells, both in vitro and in vivo. This review will summarize current progress in developing this technology, and discuss the potential impact of HITI-based gene-correction therapies.



Li J, Zhang B, Ren Y, Gu S, Xiang Y, Du J. Intron targeting-mediated and endogenous gene integrity-maintaining knockin in zebrafish using the CRISPR/Cas9 system. Cell Res. 2015;25:634-7.



Suzuki K, Tsunekawa Y, Hernandez-Benitez R, Wu J, Zhu J, Kim EJ, et al. In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration. Nature. 2016;540:144-9.



Yao X, Wang X, Liu J, Hu X, Shi L, Shen X, et al. CRISPR/Cas9 - mediated precise targeted integration in vivo using a double cut donor with short homology arms. EBioMedicine. 2017;20:19-26.



Yao X, Wang X, Hu X, Liu Z, Liu J, Zhou H, et al. Homology-mediated end joining-based targeted integration using CRISPR/Cas9. Cell Res. 2017;27:801-14.

...

Luo J, Baranov P, Patel S, Ouyang H, Quach J, Wu F, et al. Human retinal progenitor cell transplantation preserves vision. J Biol Chem. 2014;289:6362-71.

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Martin Bosnev
Martin Bosnev edited on 2 May 2019 6:20 am
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November 13, 2017

In vivo genome editing via the HITI method as a tool for gene therapy

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 In vivo genome editing via the HITI method as a tool for gene therapy

HITI is a CRISPR/Cas9-based strategy that enables gene insertion in non-dividing cells in vivo and in vitro and has applications in gene therapy.

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