Generation of Inheritable A-to-G Transitions Using Adenine Base Editing and NG-PAM Cas9 in Arabidopsis thaliana
Towards precise genome editing, base editors have been developed by fusing catalytically compromised Cas9 with deaminase components, mediating C-to-T (cytosine base editors) or A-to-G (adenine base editors) transition. We developed a set of vectors consisting of a 5′-NG-3′ PAM-recognizing variant of...
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The American Phytopathological Society
2025-01-01
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| Series: | Molecular Plant-Microbe Interactions |
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| Online Access: | https://apsjournals.apsnet.org/doi/10.1094/MPMI-10-24-0127-TA |
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| author | Yi Yun Tan Yin Yin Liew Rachelle R. Q. Lee Baptiste Castel Nga Man Chan Wei-Lin Wan Yizhong Zhang Donghui Hu Persis Chan Sang-Tae Kim Eunyoung Chae |
| author_facet | Yi Yun Tan Yin Yin Liew Rachelle R. Q. Lee Baptiste Castel Nga Man Chan Wei-Lin Wan Yizhong Zhang Donghui Hu Persis Chan Sang-Tae Kim Eunyoung Chae |
| author_sort | Yi Yun Tan |
| collection | DOAJ |
| description | Towards precise genome editing, base editors have been developed by fusing catalytically compromised Cas9 with deaminase components, mediating C-to-T (cytosine base editors) or A-to-G (adenine base editors) transition. We developed a set of vectors consisting of a 5′-NG-3′ PAM-recognizing variant of SpCas9 with adenosine deaminases TadA7.10 or TadA8e. Using a phenotype-based screen in Arabidopsis thaliana targeting multiple PDS3 intron splice sites, we achieved up to 81% somatic A-to-G editing in primary transformants at a splice acceptor site with NGG PAM, while 35% was achieved for the same target adenine with NGA PAM. Among tested vectors, pECNUS4 (Addgene #184887), carrying TadA8e, showed the highest adenine base editor (ABE) efficiency. With pECNUS4, we recreated a naturally occurring allele of DANGEROUS MIX3 (DM3) in two generations, transgene-free, for NGC PAM. We also simultaneously base-edited four redundant DM1/SSI4 homologs, encoding nucleotide-binding leucine-rich repeat (NLR) proteins, using a single gRNA with NGA PAM targeting the conserved yet functionally crucial P-loop motif of NLR proteins. We found fixation of A-to-G in three NLR genes for all three possible adenine sites within base-editing window 3-9, as the edited genes segregate in T2. Multigene targeting succeeded in rescuing the previously reported autoimmune phenotype in two generations. Mediating desired ABE on seven NLR genes simultaneously was successful as well; above 77% editing was achieved in six of the seven possible targets in a T1 plant, with the remaining having a moderately high (32%) editing. ABE application to specifically inactivate functional motifs is anticipated to expedite the discovery of novel roles for proteins. [Figure: see text] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY 4.0 International license. |
| format | Article |
| id | doaj-art-c9b08c6e058f45e9958a8166b68efa79 |
| institution | OA Journals |
| issn | 0894-0282 1943-7706 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | The American Phytopathological Society |
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| series | Molecular Plant-Microbe Interactions |
| spelling | doaj-art-c9b08c6e058f45e9958a8166b68efa792025-08-20T01:58:00ZengThe American Phytopathological SocietyMolecular Plant-Microbe Interactions0894-02821943-77062025-01-01381304210.1094/MPMI-10-24-0127-TAGeneration of Inheritable A-to-G Transitions Using Adenine Base Editing and NG-PAM Cas9 in Arabidopsis thalianaYi Yun Tan0Yin Yin Liew1Rachelle R. Q. Lee2Baptiste Castel3Nga Man Chan4Wei-Lin Wan5Yizhong Zhang6Donghui Hu7Persis Chan8Sang-Tae Kim9Eunyoung Chae10Department of Biological Sciences, National University of Singapore, 117558, SingaporeDepartment of Biological Sciences, National University of Singapore, 117558, SingaporeDepartment of Biological Sciences, National University of Singapore, 117558, SingaporeDepartment of Biological Sciences, National University of Singapore, 117558, SingaporeDepartment of Biological Sciences, National University of Singapore, 117558, SingaporeDepartment of Biological Sciences, National University of Singapore, 117558, SingaporeDepartment of Biological Sciences, National University of Singapore, 117558, SingaporeDepartment of Biological Sciences, National University of Singapore, 117558, SingaporeDepartment of Biological Sciences, National University of Singapore, 117558, SingaporeDepartment of Medical & Biological Sciences, The Catholic University of Korea, Bucheon 14662, Republic of KoreaDepartment of Biological Sciences, National University of Singapore, 117558, SingaporeTowards precise genome editing, base editors have been developed by fusing catalytically compromised Cas9 with deaminase components, mediating C-to-T (cytosine base editors) or A-to-G (adenine base editors) transition. We developed a set of vectors consisting of a 5′-NG-3′ PAM-recognizing variant of SpCas9 with adenosine deaminases TadA7.10 or TadA8e. Using a phenotype-based screen in Arabidopsis thaliana targeting multiple PDS3 intron splice sites, we achieved up to 81% somatic A-to-G editing in primary transformants at a splice acceptor site with NGG PAM, while 35% was achieved for the same target adenine with NGA PAM. Among tested vectors, pECNUS4 (Addgene #184887), carrying TadA8e, showed the highest adenine base editor (ABE) efficiency. With pECNUS4, we recreated a naturally occurring allele of DANGEROUS MIX3 (DM3) in two generations, transgene-free, for NGC PAM. We also simultaneously base-edited four redundant DM1/SSI4 homologs, encoding nucleotide-binding leucine-rich repeat (NLR) proteins, using a single gRNA with NGA PAM targeting the conserved yet functionally crucial P-loop motif of NLR proteins. We found fixation of A-to-G in three NLR genes for all three possible adenine sites within base-editing window 3-9, as the edited genes segregate in T2. Multigene targeting succeeded in rescuing the previously reported autoimmune phenotype in two generations. Mediating desired ABE on seven NLR genes simultaneously was successful as well; above 77% editing was achieved in six of the seven possible targets in a T1 plant, with the remaining having a moderately high (32%) editing. ABE application to specifically inactivate functional motifs is anticipated to expedite the discovery of novel roles for proteins. [Figure: see text] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.https://apsjournals.apsnet.org/doi/10.1094/MPMI-10-24-0127-TAadenine deaminaseCRISPR/Cas9genome editingmultigene editingNLRplant biotechnology |
| spellingShingle | Yi Yun Tan Yin Yin Liew Rachelle R. Q. Lee Baptiste Castel Nga Man Chan Wei-Lin Wan Yizhong Zhang Donghui Hu Persis Chan Sang-Tae Kim Eunyoung Chae Generation of Inheritable A-to-G Transitions Using Adenine Base Editing and NG-PAM Cas9 in Arabidopsis thaliana Molecular Plant-Microbe Interactions adenine deaminase CRISPR/Cas9 genome editing multigene editing NLR plant biotechnology |
| title | Generation of Inheritable A-to-G Transitions Using Adenine Base Editing and NG-PAM Cas9 in Arabidopsis thaliana |
| title_full | Generation of Inheritable A-to-G Transitions Using Adenine Base Editing and NG-PAM Cas9 in Arabidopsis thaliana |
| title_fullStr | Generation of Inheritable A-to-G Transitions Using Adenine Base Editing and NG-PAM Cas9 in Arabidopsis thaliana |
| title_full_unstemmed | Generation of Inheritable A-to-G Transitions Using Adenine Base Editing and NG-PAM Cas9 in Arabidopsis thaliana |
| title_short | Generation of Inheritable A-to-G Transitions Using Adenine Base Editing and NG-PAM Cas9 in Arabidopsis thaliana |
| title_sort | generation of inheritable a to g transitions using adenine base editing and ng pam cas9 in arabidopsis thaliana |
| topic | adenine deaminase CRISPR/Cas9 genome editing multigene editing NLR plant biotechnology |
| url | https://apsjournals.apsnet.org/doi/10.1094/MPMI-10-24-0127-TA |
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