Polymer-stabilized Cas9 nanoparticles and modified repair templates increase genome editing efficiency
Versatile and precise genome modifications are needed to create a wider range of adoptive cellular therapies 1 – 5 . Here we report two improvements that increase the efficiency of CRISPR–Cas9-based genome editing in clinically relevant primary cell types. Truncated Cas9 target sequences (tCTSs) add...
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| Veröffentlicht in: | Nature biotechnology 2020-01, Vol.38 (1), p.44-49 |
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| creator | Nguyen, David N. Roth, Theodore L. Li, P. Jonathan Chen, Peixin Amy Apathy, Ryan Mamedov, Murad R. Vo, Linda T. Tobin, Victoria R. Goodman, Daniel Shifrut, Eric Bluestone, Jeffrey A. Puck, Jennifer M. Szoka, Francis C. Marson, Alexander |
| description | Versatile and precise genome modifications are needed to create a wider range of adoptive cellular therapies
1
–
5
. Here we report two improvements that increase the efficiency of CRISPR–Cas9-based genome editing in clinically relevant primary cell types. Truncated Cas9 target sequences (tCTSs) added at the ends of the homology-directed repair (HDR) template interact with Cas9 ribonucleoproteins (RNPs) to shuttle the template to the nucleus, enhancing HDR efficiency approximately two- to fourfold. Furthermore, stabilizing Cas9 RNPs into nanoparticles with polyglutamic acid further improves editing efficiency by approximately twofold, reduces toxicity, and enables lyophilized storage without loss of activity. Combining the two improvements increases gene targeting efficiency even at reduced HDR template doses, yielding approximately two to six times as many viable edited cells across multiple genomic loci in diverse cell types, such as bulk (CD3
+
) T cells, CD8
+
T cells, CD4
+
T cells, regulatory T cells (Tregs), γδ T cells, B cells, natural killer cells, and primary and induced pluripotent stem cell-derived
6
hematopoietic stem progenitor cells (HSPCs).
Precise genome editing is made more efficient by stabilizing Cas9 and enhancing shuttling to the nucleus. |
| doi_str_mv | 10.1038/s41587-019-0325-6 |
| format | Article |
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1
–
5
. Here we report two improvements that increase the efficiency of CRISPR–Cas9-based genome editing in clinically relevant primary cell types. Truncated Cas9 target sequences (tCTSs) added at the ends of the homology-directed repair (HDR) template interact with Cas9 ribonucleoproteins (RNPs) to shuttle the template to the nucleus, enhancing HDR efficiency approximately two- to fourfold. Furthermore, stabilizing Cas9 RNPs into nanoparticles with polyglutamic acid further improves editing efficiency by approximately twofold, reduces toxicity, and enables lyophilized storage without loss of activity. Combining the two improvements increases gene targeting efficiency even at reduced HDR template doses, yielding approximately two to six times as many viable edited cells across multiple genomic loci in diverse cell types, such as bulk (CD3
+
) T cells, CD8
+
T cells, CD4
+
T cells, regulatory T cells (Tregs), γδ T cells, B cells, natural killer cells, and primary and induced pluripotent stem cell-derived
6
hematopoietic stem progenitor cells (HSPCs).
Precise genome editing is made more efficient by stabilizing Cas9 and enhancing shuttling to the nucleus.</description><identifier>ISSN: 1087-0156</identifier><identifier>EISSN: 1546-1696</identifier><identifier>DOI: 10.1038/s41587-019-0325-6</identifier><identifier>PMID: 31819258</identifier><language>eng</language><publisher>New York: Nature Publishing Group US</publisher><subject>631/1647/1511 ; 631/1647/1513/1967/3196 ; 631/61/201/2110 ; 631/61/350/354 ; Adult ; Agriculture ; Bioinformatics ; Biomedical and Life Sciences ; Biomedical Engineering/Biotechnology ; Biomedicine ; Biotechnology ; CD3 antigen ; CD4 antigen ; CD8 antigen ; CRISPR ; CRISPR-Associated Protein 9 - metabolism ; Editing ; Efficiency ; Gene Editing ; Gene sequencing ; Gene targeting ; Genetic engineering ; Genome editing ; Genomes ; Genomics ; Hematopoietic stem cells ; Homology ; Humans ; Immunoregulation ; Letter ; Life Sciences ; Lymphocytes ; Lymphocytes B ; Lymphocytes T ; Methods ; Nanoparticles ; Nanoparticles - chemistry ; Natural killer cells ; Nuclei (cytology) ; Pluripotency ; Polymer industry ; Polymers ; Polymers - chemistry ; Progenitor cells ; Protein Stability ; Repair ; Ribonucleoproteins ; RNA, Guide, CRISPR-Cas Systems - metabolism ; Stem cells ; Toxicity</subject><ispartof>Nature biotechnology, 2020-01, Vol.38 (1), p.44-49</ispartof><rights>The Author(s), under exclusive licence to Springer Nature America, Inc. 2019</rights><rights>COPYRIGHT 2020 Nature Publishing Group</rights><rights>COPYRIGHT 2020 Nature Publishing Group</rights><rights>2019© The Author(s), under exclusive licence to Springer Nature America, Inc. 2019</rights><rights>The Author(s), under exclusive licence to Springer Nature America, Inc. 2019.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c767t-4aff7aacc37565fc5bad950c9752b07f09f58d96e47621d67af4affc194d14573</citedby><cites>FETCH-LOGICAL-c767t-4aff7aacc37565fc5bad950c9752b07f09f58d96e47621d67af4affc194d14573</cites><orcidid>0000-0001-6808-2717 ; 0000-0001-8793-7848 ; 0000-0002-1543-3330 ; 0000-0002-5204-6401 ; 0000-0002-2734-5776 ; 0000-0002-6236-0621 ; 0000-0001-6827-0128</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1038/s41587-019-0325-6$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1038/s41587-019-0325-6$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>230,314,776,780,881,27901,27902,41464,42533,51294</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/31819258$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Nguyen, David N.</creatorcontrib><creatorcontrib>Roth, Theodore L.</creatorcontrib><creatorcontrib>Li, P. Jonathan</creatorcontrib><creatorcontrib>Chen, Peixin Amy</creatorcontrib><creatorcontrib>Apathy, Ryan</creatorcontrib><creatorcontrib>Mamedov, Murad R.</creatorcontrib><creatorcontrib>Vo, Linda T.</creatorcontrib><creatorcontrib>Tobin, Victoria R.</creatorcontrib><creatorcontrib>Goodman, Daniel</creatorcontrib><creatorcontrib>Shifrut, Eric</creatorcontrib><creatorcontrib>Bluestone, Jeffrey A.</creatorcontrib><creatorcontrib>Puck, Jennifer M.</creatorcontrib><creatorcontrib>Szoka, Francis C.</creatorcontrib><creatorcontrib>Marson, Alexander</creatorcontrib><title>Polymer-stabilized Cas9 nanoparticles and modified repair templates increase genome editing efficiency</title><title>Nature biotechnology</title><addtitle>Nat Biotechnol</addtitle><addtitle>Nat Biotechnol</addtitle><description>Versatile and precise genome modifications are needed to create a wider range of adoptive cellular therapies
1
–
5
. Here we report two improvements that increase the efficiency of CRISPR–Cas9-based genome editing in clinically relevant primary cell types. Truncated Cas9 target sequences (tCTSs) added at the ends of the homology-directed repair (HDR) template interact with Cas9 ribonucleoproteins (RNPs) to shuttle the template to the nucleus, enhancing HDR efficiency approximately two- to fourfold. Furthermore, stabilizing Cas9 RNPs into nanoparticles with polyglutamic acid further improves editing efficiency by approximately twofold, reduces toxicity, and enables lyophilized storage without loss of activity. Combining the two improvements increases gene targeting efficiency even at reduced HDR template doses, yielding approximately two to six times as many viable edited cells across multiple genomic loci in diverse cell types, such as bulk (CD3
+
) T cells, CD8
+
T cells, CD4
+
T cells, regulatory T cells (Tregs), γδ T cells, B cells, natural killer cells, and primary and induced pluripotent stem cell-derived
6
hematopoietic stem progenitor cells (HSPCs).
Precise genome editing is made more efficient by stabilizing Cas9 and enhancing shuttling to the nucleus.</description><subject>631/1647/1511</subject><subject>631/1647/1513/1967/3196</subject><subject>631/61/201/2110</subject><subject>631/61/350/354</subject><subject>Adult</subject><subject>Agriculture</subject><subject>Bioinformatics</subject><subject>Biomedical and Life Sciences</subject><subject>Biomedical Engineering/Biotechnology</subject><subject>Biomedicine</subject><subject>Biotechnology</subject><subject>CD3 antigen</subject><subject>CD4 antigen</subject><subject>CD8 antigen</subject><subject>CRISPR</subject><subject>CRISPR-Associated Protein 9 - metabolism</subject><subject>Editing</subject><subject>Efficiency</subject><subject>Gene Editing</subject><subject>Gene sequencing</subject><subject>Gene targeting</subject><subject>Genetic engineering</subject><subject>Genome editing</subject><subject>Genomes</subject><subject>Genomics</subject><subject>Hematopoietic stem cells</subject><subject>Homology</subject><subject>Humans</subject><subject>Immunoregulation</subject><subject>Letter</subject><subject>Life Sciences</subject><subject>Lymphocytes</subject><subject>Lymphocytes B</subject><subject>Lymphocytes T</subject><subject>Methods</subject><subject>Nanoparticles</subject><subject>Nanoparticles - chemistry</subject><subject>Natural killer cells</subject><subject>Nuclei (cytology)</subject><subject>Pluripotency</subject><subject>Polymer industry</subject><subject>Polymers</subject><subject>Polymers - chemistry</subject><subject>Progenitor cells</subject><subject>Protein Stability</subject><subject>Repair</subject><subject>Ribonucleoproteins</subject><subject>RNA, Guide, CRISPR-Cas Systems - metabolism</subject><subject>Stem cells</subject><subject>Toxicity</subject><issn>1087-0156</issn><issn>1546-1696</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2020</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>N95</sourceid><sourceid>8G5</sourceid><sourceid>BENPR</sourceid><sourceid>GUQSH</sourceid><sourceid>M2O</sourceid><recordid>eNqNkm1r1TAYhosobh79AX6RgiAIdiZtkzRfhHHwZTCY-PY1pOmTLqNNzpJUPP56Uzu3FZxIIAm5r_tJyHNn2VOMjjCqmtehxqRhBcK8QFVJCnovO8SkpgWmnN5Pe_RbJfQgexTCBUKI1pQ-zA4q3GBekuYw0x_dsB_BFyHK1gzmJ3T5VgaeW2ndTvpo1AAhl7bLR9cZbZLuYSeNzyOMu0HGpBqrPMgAeQ_WjZBDZ6KxfQ5aG2XAqv3j7IGWQ4AnV-sm-_ru7Zfth-L07P3J9vi0UIyyWNRSayalUhUjlGhFWtlxghRnpGwR04hr0nScQs1oiTvKpJ4tCvO6wzVh1SZ7s9TdTe0InQIbvRzEzptR-r1w0oi1Ys256N13QTmpq_Spm-z5VQHvLicIUVy4ydv0ZlGmSxkv0_gnVdUVQoRzekP1cgBhrHbpSjWaoMQxrTkpaVnN1NFfqDQ6GI1yFrRJ5yvDi5UhMRF-xF5OIYg1-PJu8OTzp_9nz76t2Ve32HYKxkJIUzD9eQyLZYXjBVfeheBBX7cDIzHnWCw5FinHYs6xmD3Pbvfx2vEnuAkoFyAkyfbgbzpwd9Vf2A77Zw</recordid><startdate>20200101</startdate><enddate>20200101</enddate><creator>Nguyen, David N.</creator><creator>Roth, Theodore L.</creator><creator>Li, P. 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Jonathan ; Chen, Peixin Amy ; Apathy, Ryan ; Mamedov, Murad R. ; Vo, Linda T. ; Tobin, Victoria R. ; Goodman, Daniel ; Shifrut, Eric ; Bluestone, Jeffrey A. ; Puck, Jennifer M. ; Szoka, Francis C. ; Marson, Alexander</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c767t-4aff7aacc37565fc5bad950c9752b07f09f58d96e47621d67af4affc194d14573</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2020</creationdate><topic>631/1647/1511</topic><topic>631/1647/1513/1967/3196</topic><topic>631/61/201/2110</topic><topic>631/61/350/354</topic><topic>Adult</topic><topic>Agriculture</topic><topic>Bioinformatics</topic><topic>Biomedical and Life Sciences</topic><topic>Biomedical Engineering/Biotechnology</topic><topic>Biomedicine</topic><topic>Biotechnology</topic><topic>CD3 antigen</topic><topic>CD4 antigen</topic><topic>CD8 antigen</topic><topic>CRISPR</topic><topic>CRISPR-Associated Protein 9 - metabolism</topic><topic>Editing</topic><topic>Efficiency</topic><topic>Gene Editing</topic><topic>Gene sequencing</topic><topic>Gene targeting</topic><topic>Genetic engineering</topic><topic>Genome editing</topic><topic>Genomes</topic><topic>Genomics</topic><topic>Hematopoietic stem cells</topic><topic>Homology</topic><topic>Humans</topic><topic>Immunoregulation</topic><topic>Letter</topic><topic>Life Sciences</topic><topic>Lymphocytes</topic><topic>Lymphocytes B</topic><topic>Lymphocytes T</topic><topic>Methods</topic><topic>Nanoparticles</topic><topic>Nanoparticles - chemistry</topic><topic>Natural killer cells</topic><topic>Nuclei (cytology)</topic><topic>Pluripotency</topic><topic>Polymer industry</topic><topic>Polymers</topic><topic>Polymers - chemistry</topic><topic>Progenitor cells</topic><topic>Protein Stability</topic><topic>Repair</topic><topic>Ribonucleoproteins</topic><topic>RNA, Guide, CRISPR-Cas Systems - metabolism</topic><topic>Stem cells</topic><topic>Toxicity</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Nguyen, David N.</creatorcontrib><creatorcontrib>Roth, Theodore L.</creatorcontrib><creatorcontrib>Li, P. 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Jonathan</au><au>Chen, Peixin Amy</au><au>Apathy, Ryan</au><au>Mamedov, Murad R.</au><au>Vo, Linda T.</au><au>Tobin, Victoria R.</au><au>Goodman, Daniel</au><au>Shifrut, Eric</au><au>Bluestone, Jeffrey A.</au><au>Puck, Jennifer M.</au><au>Szoka, Francis C.</au><au>Marson, Alexander</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Polymer-stabilized Cas9 nanoparticles and modified repair templates increase genome editing efficiency</atitle><jtitle>Nature biotechnology</jtitle><stitle>Nat Biotechnol</stitle><addtitle>Nat Biotechnol</addtitle><date>2020-01-01</date><risdate>2020</risdate><volume>38</volume><issue>1</issue><spage>44</spage><epage>49</epage><pages>44-49</pages><issn>1087-0156</issn><eissn>1546-1696</eissn><abstract>Versatile and precise genome modifications are needed to create a wider range of adoptive cellular therapies
1
–
5
. Here we report two improvements that increase the efficiency of CRISPR–Cas9-based genome editing in clinically relevant primary cell types. Truncated Cas9 target sequences (tCTSs) added at the ends of the homology-directed repair (HDR) template interact with Cas9 ribonucleoproteins (RNPs) to shuttle the template to the nucleus, enhancing HDR efficiency approximately two- to fourfold. Furthermore, stabilizing Cas9 RNPs into nanoparticles with polyglutamic acid further improves editing efficiency by approximately twofold, reduces toxicity, and enables lyophilized storage without loss of activity. Combining the two improvements increases gene targeting efficiency even at reduced HDR template doses, yielding approximately two to six times as many viable edited cells across multiple genomic loci in diverse cell types, such as bulk (CD3
+
) T cells, CD8
+
T cells, CD4
+
T cells, regulatory T cells (Tregs), γδ T cells, B cells, natural killer cells, and primary and induced pluripotent stem cell-derived
6
hematopoietic stem progenitor cells (HSPCs).
Precise genome editing is made more efficient by stabilizing Cas9 and enhancing shuttling to the nucleus.</abstract><cop>New York</cop><pub>Nature Publishing Group US</pub><pmid>31819258</pmid><doi>10.1038/s41587-019-0325-6</doi><tpages>6</tpages><orcidid>https://orcid.org/0000-0001-6808-2717</orcidid><orcidid>https://orcid.org/0000-0001-8793-7848</orcidid><orcidid>https://orcid.org/0000-0002-1543-3330</orcidid><orcidid>https://orcid.org/0000-0002-5204-6401</orcidid><orcidid>https://orcid.org/0000-0002-2734-5776</orcidid><orcidid>https://orcid.org/0000-0002-6236-0621</orcidid><orcidid>https://orcid.org/0000-0001-6827-0128</orcidid><oa>free_for_read</oa></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 1087-0156 |
| ispartof | Nature biotechnology, 2020-01, Vol.38 (1), p.44-49 |
| issn | 1087-0156 1546-1696 |
| language | eng |
| recordid | cdi_pubmedcentral_primary_oai_pubmedcentral_nih_gov_6954310 |
| source | MEDLINE; Nature; SpringerLink Journals - AutoHoldings |
| subjects | 631/1647/1511 631/1647/1513/1967/3196 631/61/201/2110 631/61/350/354 Adult Agriculture Bioinformatics Biomedical and Life Sciences Biomedical Engineering/Biotechnology Biomedicine Biotechnology CD3 antigen CD4 antigen CD8 antigen CRISPR CRISPR-Associated Protein 9 - metabolism Editing Efficiency Gene Editing Gene sequencing Gene targeting Genetic engineering Genome editing Genomes Genomics Hematopoietic stem cells Homology Humans Immunoregulation Letter Life Sciences Lymphocytes Lymphocytes B Lymphocytes T Methods Nanoparticles Nanoparticles - chemistry Natural killer cells Nuclei (cytology) Pluripotency Polymer industry Polymers Polymers - chemistry Progenitor cells Protein Stability Repair Ribonucleoproteins RNA, Guide, CRISPR-Cas Systems - metabolism Stem cells Toxicity |
| title | Polymer-stabilized Cas9 nanoparticles and modified repair templates increase genome editing efficiency |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-02-08T00%3A26%3A46IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-gale_pubme&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Polymer-stabilized%20Cas9%20nanoparticles%20and%20modified%20repair%20templates%20increase%20genome%20editing%20efficiency&rft.jtitle=Nature%20biotechnology&rft.au=Nguyen,%20David%20N.&rft.date=2020-01-01&rft.volume=38&rft.issue=1&rft.spage=44&rft.epage=49&rft.pages=44-49&rft.issn=1087-0156&rft.eissn=1546-1696&rft_id=info:doi/10.1038/s41587-019-0325-6&rft_dat=%3Cgale_pubme%3EA649526236%3C/gale_pubme%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=2343005996&rft_id=info:pmid/31819258&rft_galeid=A649526236&rfr_iscdi=true |