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Engineering Tripartite Gene Editing Machinery for Highly Efficient Non-Viral Targeted Genome Integration
Non-viral DNA donor template has been widely used for targeted genomic integration by homologous recombination (HR). This process has become more efficient with RNA guided endonuclease editor system such as CRISPR/Cas9. Circular single stranded DNA (cssDNA) has been harnessed previously as a genome...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Journal Experts
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10635301/ https://www.ncbi.nlm.nih.gov/pubmed/37961210 http://dx.doi.org/10.21203/rs.3.rs-3365585/v1 |
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author | Nam, Hangu Xie, Keqiang Majumdar, Ishita Yang, Shaobo Starzyk, Jakob Lee, Danna Shan, Richard Li, Jiahe Wu, Hao |
author_facet | Nam, Hangu Xie, Keqiang Majumdar, Ishita Yang, Shaobo Starzyk, Jakob Lee, Danna Shan, Richard Li, Jiahe Wu, Hao |
author_sort | Nam, Hangu |
collection | PubMed |
description | Non-viral DNA donor template has been widely used for targeted genomic integration by homologous recombination (HR). This process has become more efficient with RNA guided endonuclease editor system such as CRISPR/Cas9. Circular single stranded DNA (cssDNA) has been harnessed previously as a genome engineering catalyst (GATALYST) for efficient and safe targeted gene knock-in. Here we developed enGager, a system with enhanced GATALYST associated genome editor, comprising a set of novel genome editors in which the integration efficiency of a circular single-stranded (css) donor DNA is elevated by directly tethering of the cssDNA to a nuclear-localized Cas9 fused with ssDNA binding peptides. Improvements in site-directed genomic integration and expression of a knocked-in DNA encoding GFP were observed at multiple genomic loci in multiple cell lines. The enhancement of integration efficiency, compared to unfused Cas9 editors, ranges from 1.5- to more than 6-fold, with the enhancement most pronounced for transgenes of > 4Kb in length in primary cells. enGager-enhanced genome integration prefers ssDNA donors which, unlike traditional dsDNA donors, are not concatemerized or rearranged prior to and during integration Using an enGager fused to an optimized cssDNA binding peptide, exceptionally efficient, targeted integration of the chimeric antigen receptor (CAR) transgene was achieved in 33% of primary human T cells. Enhanced anti-tumor function of these CAR-T primary cells demonstrated the functional competence of the transgenes. The ‘tripartite editors with ssDNA optimized genome engineering’ (TESOGENASE(™)) systems help address the efficacy needs for therapeutic gene modification while avoiding the safety and payload size limitations of viral vectors currently used for CAR-T engineering. |
format | Online Article Text |
id | pubmed-10635301 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | American Journal Experts |
record_format | MEDLINE/PubMed |
spelling | pubmed-106353012023-11-13 Engineering Tripartite Gene Editing Machinery for Highly Efficient Non-Viral Targeted Genome Integration Nam, Hangu Xie, Keqiang Majumdar, Ishita Yang, Shaobo Starzyk, Jakob Lee, Danna Shan, Richard Li, Jiahe Wu, Hao Res Sq Article Non-viral DNA donor template has been widely used for targeted genomic integration by homologous recombination (HR). This process has become more efficient with RNA guided endonuclease editor system such as CRISPR/Cas9. Circular single stranded DNA (cssDNA) has been harnessed previously as a genome engineering catalyst (GATALYST) for efficient and safe targeted gene knock-in. Here we developed enGager, a system with enhanced GATALYST associated genome editor, comprising a set of novel genome editors in which the integration efficiency of a circular single-stranded (css) donor DNA is elevated by directly tethering of the cssDNA to a nuclear-localized Cas9 fused with ssDNA binding peptides. Improvements in site-directed genomic integration and expression of a knocked-in DNA encoding GFP were observed at multiple genomic loci in multiple cell lines. The enhancement of integration efficiency, compared to unfused Cas9 editors, ranges from 1.5- to more than 6-fold, with the enhancement most pronounced for transgenes of > 4Kb in length in primary cells. enGager-enhanced genome integration prefers ssDNA donors which, unlike traditional dsDNA donors, are not concatemerized or rearranged prior to and during integration Using an enGager fused to an optimized cssDNA binding peptide, exceptionally efficient, targeted integration of the chimeric antigen receptor (CAR) transgene was achieved in 33% of primary human T cells. Enhanced anti-tumor function of these CAR-T primary cells demonstrated the functional competence of the transgenes. The ‘tripartite editors with ssDNA optimized genome engineering’ (TESOGENASE(™)) systems help address the efficacy needs for therapeutic gene modification while avoiding the safety and payload size limitations of viral vectors currently used for CAR-T engineering. American Journal Experts 2023-10-23 /pmc/articles/PMC10635301/ /pubmed/37961210 http://dx.doi.org/10.21203/rs.3.rs-3365585/v1 Text en https://creativecommons.org/licenses/by/4.0/This work is licensed under a Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/) , which allows reusers to distribute, remix, adapt, and build upon the material in any medium or format, so long as attribution is given to the creator. The license allows for commercial use. |
spellingShingle | Article Nam, Hangu Xie, Keqiang Majumdar, Ishita Yang, Shaobo Starzyk, Jakob Lee, Danna Shan, Richard Li, Jiahe Wu, Hao Engineering Tripartite Gene Editing Machinery for Highly Efficient Non-Viral Targeted Genome Integration |
title | Engineering Tripartite Gene Editing Machinery for Highly Efficient Non-Viral Targeted Genome Integration |
title_full | Engineering Tripartite Gene Editing Machinery for Highly Efficient Non-Viral Targeted Genome Integration |
title_fullStr | Engineering Tripartite Gene Editing Machinery for Highly Efficient Non-Viral Targeted Genome Integration |
title_full_unstemmed | Engineering Tripartite Gene Editing Machinery for Highly Efficient Non-Viral Targeted Genome Integration |
title_short | Engineering Tripartite Gene Editing Machinery for Highly Efficient Non-Viral Targeted Genome Integration |
title_sort | engineering tripartite gene editing machinery for highly efficient non-viral targeted genome integration |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10635301/ https://www.ncbi.nlm.nih.gov/pubmed/37961210 http://dx.doi.org/10.21203/rs.3.rs-3365585/v1 |
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