Improvement of the LbCas12a-crRNA System for Efficient Gene Targeting in Tomato
Plant gene targeting (GT) can be utilized to precisely replace up to several kilobases of a plant genome. Recent studies using the powerful clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) nucleases significantly improved plant GT efficiency. However, GT...
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Frontiers Media S.A.
2021-08-01
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| Series: | Frontiers in Plant Science |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fpls.2021.722552/full |
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| author | Tien Van Vu Tien Van Vu Duong Thi Hai Doan Mil Thi Tran Mil Thi Tran Yeon Woo Sung Young Jong Song Jae-Yean Kim Jae-Yean Kim |
| author_facet | Tien Van Vu Tien Van Vu Duong Thi Hai Doan Mil Thi Tran Mil Thi Tran Yeon Woo Sung Young Jong Song Jae-Yean Kim Jae-Yean Kim |
| author_sort | Tien Van Vu |
| collection | DOAJ |
| description | Plant gene targeting (GT) can be utilized to precisely replace up to several kilobases of a plant genome. Recent studies using the powerful clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) nucleases significantly improved plant GT efficiency. However, GT for loci without associated selection markers is still inefficient. We previously utilized Lachnospiraceae bacterium Cas12a (LbCas12a) in combination with a replicon for tomato GT and obtained high GT efficiency with some selection markers. In this study, we advance our GT system by inhibiting the cNHEJ pathway with small chemical molecules such as NU7441. Further optimization of the GT is also possible with the treatment of silver nitrate possibly via its pronounced actions in ethylene inhibition and polyamine production. Importantly, the GT efficiency is significantly enhanced with the use of a temperature-tolerant LbCas12a (ttLbCas12a) that is capable of performing target cleavage even at low temperatures. Targeted deep sequencing, as well as conventional methods, are used for the assessment of the editing efficiency at both cell and plant levels. Our work demonstrates the significance of the selection of gene scissors, the appropriate design and number of LbCas12a crRNAs, the use of chemical treatments, and the establishment of favorable experimental conditions for further enhancement of plant HDR to enable efficient GT in tomato. |
| first_indexed | 2024-12-16T12:32:18Z |
| format | Article |
| id | doaj.art-8afe0e0377c147d586fa4075b007042c |
| institution | Directory Open Access Journal |
| issn | 1664-462X |
| language | English |
| last_indexed | 2024-12-16T12:32:18Z |
| publishDate | 2021-08-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Plant Science |
| spelling | doaj.art-8afe0e0377c147d586fa4075b007042c2022-12-21T22:31:40ZengFrontiers Media S.A.Frontiers in Plant Science1664-462X2021-08-011210.3389/fpls.2021.722552722552Improvement of the LbCas12a-crRNA System for Efficient Gene Targeting in TomatoTien Van Vu0Tien Van Vu1Duong Thi Hai Doan2Mil Thi Tran3Mil Thi Tran4Yeon Woo Sung5Young Jong Song6Jae-Yean Kim7Jae-Yean Kim8Division of Applied Life Science (BK21 FOUR Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju, South KoreaNational Key Laboratory for Plant Cell Biotechnology, Agricultural Genetics Institute, Hanoi, VietnamDivision of Applied Life Science (BK21 FOUR Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju, South KoreaDivision of Applied Life Science (BK21 FOUR Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju, South KoreaCrop Science and Rural Development Division, College of Agriculture, Bac Lieu University, Bạc Liêu, VietnamDivision of Applied Life Science (BK21 FOUR Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju, South KoreaDivision of Applied Life Science (BK21 FOUR Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju, South KoreaDivision of Applied Life Science (BK21 FOUR Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju, South KoreaDivision of Life Science, Gyeongsang National University, Jinju, South KoreaPlant gene targeting (GT) can be utilized to precisely replace up to several kilobases of a plant genome. Recent studies using the powerful clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) nucleases significantly improved plant GT efficiency. However, GT for loci without associated selection markers is still inefficient. We previously utilized Lachnospiraceae bacterium Cas12a (LbCas12a) in combination with a replicon for tomato GT and obtained high GT efficiency with some selection markers. In this study, we advance our GT system by inhibiting the cNHEJ pathway with small chemical molecules such as NU7441. Further optimization of the GT is also possible with the treatment of silver nitrate possibly via its pronounced actions in ethylene inhibition and polyamine production. Importantly, the GT efficiency is significantly enhanced with the use of a temperature-tolerant LbCas12a (ttLbCas12a) that is capable of performing target cleavage even at low temperatures. Targeted deep sequencing, as well as conventional methods, are used for the assessment of the editing efficiency at both cell and plant levels. Our work demonstrates the significance of the selection of gene scissors, the appropriate design and number of LbCas12a crRNAs, the use of chemical treatments, and the establishment of favorable experimental conditions for further enhancement of plant HDR to enable efficient GT in tomato.https://www.frontiersin.org/articles/10.3389/fpls.2021.722552/fullLbCas12agene targetinggene editinghomology-directed repairSpCas9geminiviral replicon |
| spellingShingle | Tien Van Vu Tien Van Vu Duong Thi Hai Doan Mil Thi Tran Mil Thi Tran Yeon Woo Sung Young Jong Song Jae-Yean Kim Jae-Yean Kim Improvement of the LbCas12a-crRNA System for Efficient Gene Targeting in Tomato Frontiers in Plant Science LbCas12a gene targeting gene editing homology-directed repair SpCas9 geminiviral replicon |
| title | Improvement of the LbCas12a-crRNA System for Efficient Gene Targeting in Tomato |
| title_full | Improvement of the LbCas12a-crRNA System for Efficient Gene Targeting in Tomato |
| title_fullStr | Improvement of the LbCas12a-crRNA System for Efficient Gene Targeting in Tomato |
| title_full_unstemmed | Improvement of the LbCas12a-crRNA System for Efficient Gene Targeting in Tomato |
| title_short | Improvement of the LbCas12a-crRNA System for Efficient Gene Targeting in Tomato |
| title_sort | improvement of the lbcas12a crrna system for efficient gene targeting in tomato |
| topic | LbCas12a gene targeting gene editing homology-directed repair SpCas9 geminiviral replicon |
| url | https://www.frontiersin.org/articles/10.3389/fpls.2021.722552/full |
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