Dual guide RNA-mediated concurrent C&G-to-T&A and A&T-to-G&C conversions using CRISPR base editors
Base editing tools enable precise genome modifications, disease modeling, and promising gene therapy. However, many human genetic diseases are elicited by multi-nucleotide variants (MNVs) with heterogeneous substitutions at the same genomic locus. Based on the adenine and cytosine base editors, dual...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2023-01-01
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| Series: | Computational and Structural Biotechnology Journal |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2001037022006109 |
| _version_ | 1827577658431504384 |
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| author | Yuting Zhao Min Li Jie Liu Xiaowen Xue Jingli Zhong Jianxiang Lin Bo Ye Jun Chen Yunbo Qiao |
| author_facet | Yuting Zhao Min Li Jie Liu Xiaowen Xue Jingli Zhong Jianxiang Lin Bo Ye Jun Chen Yunbo Qiao |
| author_sort | Yuting Zhao |
| collection | DOAJ |
| description | Base editing tools enable precise genome modifications, disease modeling, and promising gene therapy. However, many human genetic diseases are elicited by multi-nucleotide variants (MNVs) with heterogeneous substitutions at the same genomic locus. Based on the adenine and cytosine base editors, dual base editors that can catalyze concurrent C-to-T and A-to-G editing have been developed, while simultaneous C&G-to-T&A and A&T-to-G&C conversions on the same allele have not been achieved at the desirable site. Here we propose a strategy of combining base editors with dual guide RNAs (gRNAs) that target two overlapped neighboring loci on the opposite strands, which can induce simultaneous C&G-to-T&A and A&T-to-G&C conversions within their overlapping targeting windows. Moreover, one of the paired gRNAs is mutated to perfectly match another gRNA-edited sequence, efficiently facilitating concurrent base conversions on the same allele. To further expand the targeting scopes, PAMless SpRY Cas9-mediated base editors are combined with our optimized dual gRNAs system to induce expected concurrent base editing and to install neighboring pathogenic MNVs in TP53 in cancer cells. In addition, more complex mutation types can be achieved by integrating dual base editors and our dual gRNAs strategy. Thus, we establish a general strategy to efficiently induce MNVs in human genome, helping to dissect the functions of pathogenic MNVs with multifarious types. |
| first_indexed | 2024-03-08T21:30:56Z |
| format | Article |
| id | doaj.art-3751f610acf24dcf892aa48a3987aeac |
| institution | Directory Open Access Journal |
| issn | 2001-0370 |
| language | English |
| last_indexed | 2024-03-08T21:30:56Z |
| publishDate | 2023-01-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Computational and Structural Biotechnology Journal |
| spelling | doaj.art-3751f610acf24dcf892aa48a3987aeac2023-12-21T07:30:41ZengElsevierComputational and Structural Biotechnology Journal2001-03702023-01-0121856868Dual guide RNA-mediated concurrent C&G-to-T&A and A&T-to-G&C conversions using CRISPR base editorsYuting Zhao0Min Li1Jie Liu2Xiaowen Xue3Jingli Zhong4Jianxiang Lin5Bo Ye6Jun Chen7Yunbo Qiao8Precise Genome Engineering Center, School of Life Sciences, Guangzhou University, Guangzhou 510006, ChinaPrecise Genome Engineering Center, School of Life Sciences, Guangzhou University, Guangzhou 510006, ChinaGuangzhou Medical University, Guangzhou 511495, ChinaPrecise Genome Engineering Center, School of Life Sciences, Guangzhou University, Guangzhou 510006, ChinaPrecise Genome Engineering Center, School of Life Sciences, Guangzhou University, Guangzhou 510006, ChinaPrecise Genome Engineering Center, School of Life Sciences, Guangzhou University, Guangzhou 510006, ChinaDepartment of Thoracic Surgery, Shanghai Chest Hospital, Shanghai Jiao Tong University, Shanghai 200030, China; Corresponding authors.Department of Endocrinology, Qilu Hospital, Cheeloo College of Medicine, Shandong University, Jinan 250012, China; Corresponding authors.Precise Genome Engineering Center, School of Life Sciences, Guangzhou University, Guangzhou 510006, China; Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200125, China; Shanghai Institute of Precision Medicine, Shanghai 200125, China; Corresponding author at: Precise Genome Engineering Center, School of Life Sciences, Guangzhou University, Guangzhou 510006, China.Base editing tools enable precise genome modifications, disease modeling, and promising gene therapy. However, many human genetic diseases are elicited by multi-nucleotide variants (MNVs) with heterogeneous substitutions at the same genomic locus. Based on the adenine and cytosine base editors, dual base editors that can catalyze concurrent C-to-T and A-to-G editing have been developed, while simultaneous C&G-to-T&A and A&T-to-G&C conversions on the same allele have not been achieved at the desirable site. Here we propose a strategy of combining base editors with dual guide RNAs (gRNAs) that target two overlapped neighboring loci on the opposite strands, which can induce simultaneous C&G-to-T&A and A&T-to-G&C conversions within their overlapping targeting windows. Moreover, one of the paired gRNAs is mutated to perfectly match another gRNA-edited sequence, efficiently facilitating concurrent base conversions on the same allele. To further expand the targeting scopes, PAMless SpRY Cas9-mediated base editors are combined with our optimized dual gRNAs system to induce expected concurrent base editing and to install neighboring pathogenic MNVs in TP53 in cancer cells. In addition, more complex mutation types can be achieved by integrating dual base editors and our dual gRNAs strategy. Thus, we establish a general strategy to efficiently induce MNVs in human genome, helping to dissect the functions of pathogenic MNVs with multifarious types.http://www.sciencedirect.com/science/article/pii/S2001037022006109Dual guide RNAs (gRNAs)Multi-nucleotide variant (MNV)C&G-to-T&A and A&T-to-G&C conversionsTP53SpRY |
| spellingShingle | Yuting Zhao Min Li Jie Liu Xiaowen Xue Jingli Zhong Jianxiang Lin Bo Ye Jun Chen Yunbo Qiao Dual guide RNA-mediated concurrent C&G-to-T&A and A&T-to-G&C conversions using CRISPR base editors Computational and Structural Biotechnology Journal Dual guide RNAs (gRNAs) Multi-nucleotide variant (MNV) C&G-to-T&A and A&T-to-G&C conversions TP53 SpRY |
| title | Dual guide RNA-mediated concurrent C&G-to-T&A and A&T-to-G&C conversions using CRISPR base editors |
| title_full | Dual guide RNA-mediated concurrent C&G-to-T&A and A&T-to-G&C conversions using CRISPR base editors |
| title_fullStr | Dual guide RNA-mediated concurrent C&G-to-T&A and A&T-to-G&C conversions using CRISPR base editors |
| title_full_unstemmed | Dual guide RNA-mediated concurrent C&G-to-T&A and A&T-to-G&C conversions using CRISPR base editors |
| title_short | Dual guide RNA-mediated concurrent C&G-to-T&A and A&T-to-G&C conversions using CRISPR base editors |
| title_sort | dual guide rna mediated concurrent c g to t a and a t to g c conversions using crispr base editors |
| topic | Dual guide RNAs (gRNAs) Multi-nucleotide variant (MNV) C&G-to-T&A and A&T-to-G&C conversions TP53 SpRY |
| url | http://www.sciencedirect.com/science/article/pii/S2001037022006109 |
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