A split prime editor with untethered reverse transcriptase and circular RNA template
Delivery and optimization of prime editors (PEs) have been hampered by their large size and complexity. Although split versions of genome-editing tools can reduce construct size, they require special engineering to tether the binding and catalytic domains. Here we report a split PE (sPE) in which th...
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| Veröffentlicht in: | Nature biotechnology 2022-09, Vol.40 (9), p.1388-1393 |
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| creator | Liu, Bin Dong, Xiaolong Cheng, Haoyang Zheng, Chunwei Chen, Zexiang Rodríguez, Tomás C. Liang, Shun-Qing Xue, Wen Sontheimer, Erik J. |
| description | Delivery and optimization of prime editors (PEs) have been hampered by their large size and complexity. Although split versions of genome-editing tools can reduce construct size, they require special engineering to tether the binding and catalytic domains. Here we report a split PE (sPE) in which the Cas9 nickase (nCas9) remains untethered from the reverse transcriptase (RT). The sPE showed similar efficiencies in installing precise edits as the parental unsplit PE3 and no increase in insertion–deletion (indel) byproducts. Delivery of sPE to the mouse liver with hydrodynamic injection to modify β-catenin drove tumor formation with similar efficiency as PE3. Delivery with two adeno-associated virus (AAV) vectors corrected the disease-causing mutation in a mouse model of type I tyrosinemia. Similarly, prime editing guide RNAs (pegRNAs) can be split into a single guide RNA (sgRNA) and a circular RNA RT template to increase flexibility and stability. Compared to previous sPEs, ours lacks inteins, protein–protein affinity modules and nuclease-sensitive pegRNA extensions, which increase construct complexity and might reduce efficiency. Our modular system will facilitate the delivery and optimization of PEs.
A split prime editor simplifies delivery. |
| doi_str_mv | 10.1038/s41587-022-01255-9 |
| format | Article |
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A split prime editor simplifies delivery.</description><subject>631/1647/1511</subject><subject>692/308/2056</subject><subject>Agriculture</subject><subject>Animals</subject><subject>Binding sites</subject><subject>Bioinformatics</subject><subject>Biomedical and Life Sciences</subject><subject>Biomedical Engineering/Biotechnology</subject><subject>Biomedicine</subject><subject>Biotechnology</subject><subject>Circular RNA</subject><subject>Complexity</subject><subject>CRISPR-Cas Systems</subject><subject>Deoxyribonuclease I - genetics</subject><subject>Editing</subject><subject>Efficiency</subject><subject>Gene Editing</subject><subject>Genome editing</subject><subject>Genomes</subject><subject>Insertion</subject><subject>Inteins</subject><subject>Life Sciences</subject><subject>Medical schools</subject><subject>Mice</subject><subject>Modular construction</subject><subject>Modular systems</subject><subject>Mutation</subject><subject>Nuclease</subject><subject>Optimization</subject><subject>Plasmids</subject><subject>Protein structure</subject><subject>Proteins</subject><subject>Ribonucleic acid</subject><subject>RNA</subject><subject>RNA, Circular - genetics</subject><subject>RNA, Guide, CRISPR-Cas Systems</subject><subject>RNA-directed DNA polymerase</subject><subject>RNA-Directed DNA Polymerase - genetics</subject><subject>Tumors</subject><subject>Tyrosinemias - genetics</subject><subject>β-Catenin</subject><issn>1087-0156</issn><issn>1546-1696</issn><issn>1546-1696</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2022</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>8G5</sourceid><sourceid>BENPR</sourceid><sourceid>GUQSH</sourceid><sourceid>M2O</sourceid><recordid>eNp9kE1LAzEQhoMoWqt_wIMEvHhZzXd2j6X4BUVB6jlk01m7st1dk6zivze1VcGDpyTMM-9MHoROKLmghOeXQVCZ64wwlhHKpMyKHTSiUqiMqkLtpjtZl6lUB-gwhBdCiBJK7aMDLrkuCsVGaD7BoW_qiHtfrwDDoo6dx-91XOKhjRCX4GGBPbyBD4Cjt21wvu6jTS_bLrCrvRsa6_Hj_QRHWPWNjXCE9irbBDjenmP0dH01n95ms4ebu-lkljmuZcxsXkrH87SILIFIwR1zjHFHaC5KDSA4d1o7lX6Za2UFBV5VlaQ5k6KkVPAxOt_k9r57HSBEs6qDg6axLXRDMEwJzUhRcJbQsz_oSzf4Nm1nmKaM0pwrnSi2oZzvQvBQmbUW6z8MJWbt3Gycm-TcfDk3RWo63UYP5QoWPy3fkhPAN0BIpfYZ_O_sf2I_ATdLiyg</recordid><startdate>20220901</startdate><enddate>20220901</enddate><creator>Liu, Bin</creator><creator>Dong, Xiaolong</creator><creator>Cheng, Haoyang</creator><creator>Zheng, Chunwei</creator><creator>Chen, Zexiang</creator><creator>Rodríguez, Tomás C.</creator><creator>Liang, Shun-Qing</creator><creator>Xue, Wen</creator><creator>Sontheimer, Erik J.</creator><general>Nature Publishing Group US</general><general>Nature Publishing Group</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>3V.</scope><scope>7QO</scope><scope>7QP</scope><scope>7QR</scope><scope>7T7</scope><scope>7TK</scope><scope>7TM</scope><scope>7X7</scope><scope>7XB</scope><scope>88A</scope><scope>88E</scope><scope>88I</scope><scope>8AO</scope><scope>8FD</scope><scope>8FE</scope><scope>8FG</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>8G5</scope><scope>ABJCF</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BGLVJ</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>GUQSH</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>L6V</scope><scope>LK8</scope><scope>M0S</scope><scope>M1P</scope><scope>M2O</scope><scope>M2P</scope><scope>M7P</scope><scope>M7S</scope><scope>MBDVC</scope><scope>P64</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PTHSS</scope><scope>Q9U</scope><scope>RC3</scope><scope>7X8</scope><orcidid>https://orcid.org/0000-0003-3313-9853</orcidid><orcidid>https://orcid.org/0000-0002-9797-8042</orcidid><orcidid>https://orcid.org/0000-0002-0881-0310</orcidid><orcidid>https://orcid.org/0000-0002-1584-2417</orcidid><orcidid>https://orcid.org/0000-0002-8724-5427</orcidid><orcidid>https://orcid.org/0000-0002-3168-8456</orcidid></search><sort><creationdate>20220901</creationdate><title>A split prime editor with untethered reverse transcriptase and circular RNA template</title><author>Liu, Bin ; Dong, Xiaolong ; Cheng, Haoyang ; Zheng, Chunwei ; Chen, Zexiang ; Rodríguez, Tomás C. ; Liang, Shun-Qing ; Xue, Wen ; Sontheimer, Erik J.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c375t-a8b5c389965be0543c2c223c0184b7ee433c77c6158876a41e3fff518254b1143</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2022</creationdate><topic>631/1647/1511</topic><topic>692/308/2056</topic><topic>Agriculture</topic><topic>Animals</topic><topic>Binding sites</topic><topic>Bioinformatics</topic><topic>Biomedical and Life Sciences</topic><topic>Biomedical Engineering/Biotechnology</topic><topic>Biomedicine</topic><topic>Biotechnology</topic><topic>Circular RNA</topic><topic>Complexity</topic><topic>CRISPR-Cas Systems</topic><topic>Deoxyribonuclease I - genetics</topic><topic>Editing</topic><topic>Efficiency</topic><topic>Gene Editing</topic><topic>Genome editing</topic><topic>Genomes</topic><topic>Insertion</topic><topic>Inteins</topic><topic>Life Sciences</topic><topic>Medical schools</topic><topic>Mice</topic><topic>Modular construction</topic><topic>Modular systems</topic><topic>Mutation</topic><topic>Nuclease</topic><topic>Optimization</topic><topic>Plasmids</topic><topic>Protein structure</topic><topic>Proteins</topic><topic>Ribonucleic acid</topic><topic>RNA</topic><topic>RNA, Circular - genetics</topic><topic>RNA, Guide, CRISPR-Cas Systems</topic><topic>RNA-directed DNA polymerase</topic><topic>RNA-Directed DNA Polymerase - genetics</topic><topic>Tumors</topic><topic>Tyrosinemias - genetics</topic><topic>β-Catenin</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Liu, Bin</creatorcontrib><creatorcontrib>Dong, Xiaolong</creatorcontrib><creatorcontrib>Cheng, Haoyang</creatorcontrib><creatorcontrib>Zheng, Chunwei</creatorcontrib><creatorcontrib>Chen, Zexiang</creatorcontrib><creatorcontrib>Rodríguez, Tomás C.</creatorcontrib><creatorcontrib>Liang, Shun-Qing</creatorcontrib><creatorcontrib>Xue, Wen</creatorcontrib><creatorcontrib>Sontheimer, Erik J.</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>ProQuest Central (Corporate)</collection><collection>Biotechnology Research Abstracts</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>Chemoreception Abstracts</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>Neurosciences Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Biology Database (Alumni Edition)</collection><collection>Medical Database (Alumni Edition)</collection><collection>Science Database (Alumni Edition)</collection><collection>ProQuest Pharma Collection</collection><collection>Technology Research Database</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Technology Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>Research Library (Alumni Edition)</collection><collection>Materials Science & Engineering Collection</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest One Sustainability</collection><collection>ProQuest Central UK/Ireland</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Technology Collection (ProQuest)</collection><collection>Natural Science Collection</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</collection><collection>Engineering Research Database</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>Research Library Prep</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>ProQuest Engineering Collection</collection><collection>ProQuest Biological Science Collection</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Research Library</collection><collection>Science Database</collection><collection>Biological Science Database</collection><collection>Engineering Database</collection><collection>Research Library (Corporate)</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>Engineering Collection</collection><collection>ProQuest Central Basic</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Nature biotechnology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Liu, Bin</au><au>Dong, Xiaolong</au><au>Cheng, Haoyang</au><au>Zheng, Chunwei</au><au>Chen, Zexiang</au><au>Rodríguez, Tomás C.</au><au>Liang, Shun-Qing</au><au>Xue, Wen</au><au>Sontheimer, Erik J.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>A split prime editor with untethered reverse transcriptase and circular RNA template</atitle><jtitle>Nature biotechnology</jtitle><stitle>Nat Biotechnol</stitle><addtitle>Nat Biotechnol</addtitle><date>2022-09-01</date><risdate>2022</risdate><volume>40</volume><issue>9</issue><spage>1388</spage><epage>1393</epage><pages>1388-1393</pages><issn>1087-0156</issn><issn>1546-1696</issn><eissn>1546-1696</eissn><abstract>Delivery and optimization of prime editors (PEs) have been hampered by their large size and complexity. Although split versions of genome-editing tools can reduce construct size, they require special engineering to tether the binding and catalytic domains. Here we report a split PE (sPE) in which the Cas9 nickase (nCas9) remains untethered from the reverse transcriptase (RT). The sPE showed similar efficiencies in installing precise edits as the parental unsplit PE3 and no increase in insertion–deletion (indel) byproducts. Delivery of sPE to the mouse liver with hydrodynamic injection to modify β-catenin drove tumor formation with similar efficiency as PE3. Delivery with two adeno-associated virus (AAV) vectors corrected the disease-causing mutation in a mouse model of type I tyrosinemia. Similarly, prime editing guide RNAs (pegRNAs) can be split into a single guide RNA (sgRNA) and a circular RNA RT template to increase flexibility and stability. Compared to previous sPEs, ours lacks inteins, protein–protein affinity modules and nuclease-sensitive pegRNA extensions, which increase construct complexity and might reduce efficiency. Our modular system will facilitate the delivery and optimization of PEs.
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| source | MEDLINE; Springer Nature - Complete Springer Journals; Nature Journals Online |
| subjects | 631/1647/1511 692/308/2056 Agriculture Animals Binding sites Bioinformatics Biomedical and Life Sciences Biomedical Engineering/Biotechnology Biomedicine Biotechnology Circular RNA Complexity CRISPR-Cas Systems Deoxyribonuclease I - genetics Editing Efficiency Gene Editing Genome editing Genomes Insertion Inteins Life Sciences Medical schools Mice Modular construction Modular systems Mutation Nuclease Optimization Plasmids Protein structure Proteins Ribonucleic acid RNA RNA, Circular - genetics RNA, Guide, CRISPR-Cas Systems RNA-directed DNA polymerase RNA-Directed DNA Polymerase - genetics Tumors Tyrosinemias - genetics β-Catenin |
| title | A split prime editor with untethered reverse transcriptase and circular RNA template |
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