BE4max and AncBE4max Are Efficient in Germline Conversion of C:G to T:A Base Pairs in Zebrafish
The ease of use and robustness of genome editing by CRISPR/Cas9 has led to successful use of gene knockout zebrafish for disease modeling. However, it still remains a challenge to precisely edit the zebrafish genome to create single-nucleotide substitutions, which account for ~60% of human disease-c...
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| Format: | Article |
| Language: | English |
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MDPI AG
2020-07-01
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| Series: | Cells |
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| Online Access: | https://www.mdpi.com/2073-4409/9/7/1690 |
| _version_ | 1797562469488525312 |
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| author | Blake Carrington Rachel N. Weinstein Raman Sood |
| author_facet | Blake Carrington Rachel N. Weinstein Raman Sood |
| author_sort | Blake Carrington |
| collection | DOAJ |
| description | The ease of use and robustness of genome editing by CRISPR/Cas9 has led to successful use of gene knockout zebrafish for disease modeling. However, it still remains a challenge to precisely edit the zebrafish genome to create single-nucleotide substitutions, which account for ~60% of human disease-causing mutations. Recently developed base editing nucleases provide an excellent alternate to CRISPR/Cas9-mediated homology dependent repair for generation of zebrafish with point mutations. A new set of cytosine base editors, termed BE4max and AncBE4max, demonstrated improved base editing efficiency in mammalian cells but have not been evaluated in zebrafish. Therefore, we undertook this study to evaluate their efficiency in converting C:G to T:A base pairs in zebrafish by somatic and germline analysis using highly active sgRNAs to <i>twist</i> and <i>ntl</i> genes. Our data demonstrated that these improved BE4max set of plasmids provide desired base substitutions at similar efficiency and without any indels compared to the previously reported BE3 and Target-AID plasmids in zebrafish. Our data also showed that AncBE4max produces fewer incorrect and bystander edits, suggesting that it can be further improved by codon optimization of its components for use in zebrafish. |
| first_indexed | 2024-03-10T18:29:38Z |
| format | Article |
| id | doaj.art-68dba495094e44b48baf16cfe8c9f12a |
| institution | Directory Open Access Journal |
| issn | 2073-4409 |
| language | English |
| last_indexed | 2024-03-10T18:29:38Z |
| publishDate | 2020-07-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Cells |
| spelling | doaj.art-68dba495094e44b48baf16cfe8c9f12a2023-11-20T06:43:21ZengMDPI AGCells2073-44092020-07-0197169010.3390/cells9071690BE4max and AncBE4max Are Efficient in Germline Conversion of C:G to T:A Base Pairs in ZebrafishBlake Carrington0Rachel N. Weinstein1Raman Sood2Zebrafish Core, Translational and Functional Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USAZebrafish Core, Translational and Functional Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USAZebrafish Core, Translational and Functional Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USAThe ease of use and robustness of genome editing by CRISPR/Cas9 has led to successful use of gene knockout zebrafish for disease modeling. However, it still remains a challenge to precisely edit the zebrafish genome to create single-nucleotide substitutions, which account for ~60% of human disease-causing mutations. Recently developed base editing nucleases provide an excellent alternate to CRISPR/Cas9-mediated homology dependent repair for generation of zebrafish with point mutations. A new set of cytosine base editors, termed BE4max and AncBE4max, demonstrated improved base editing efficiency in mammalian cells but have not been evaluated in zebrafish. Therefore, we undertook this study to evaluate their efficiency in converting C:G to T:A base pairs in zebrafish by somatic and germline analysis using highly active sgRNAs to <i>twist</i> and <i>ntl</i> genes. Our data demonstrated that these improved BE4max set of plasmids provide desired base substitutions at similar efficiency and without any indels compared to the previously reported BE3 and Target-AID plasmids in zebrafish. Our data also showed that AncBE4max produces fewer incorrect and bystander edits, suggesting that it can be further improved by codon optimization of its components for use in zebrafish.https://www.mdpi.com/2073-4409/9/7/1690zebrafishbase editinggenome editingBE4maxAncBE4max |
| spellingShingle | Blake Carrington Rachel N. Weinstein Raman Sood BE4max and AncBE4max Are Efficient in Germline Conversion of C:G to T:A Base Pairs in Zebrafish Cells zebrafish base editing genome editing BE4max AncBE4max |
| title | BE4max and AncBE4max Are Efficient in Germline Conversion of C:G to T:A Base Pairs in Zebrafish |
| title_full | BE4max and AncBE4max Are Efficient in Germline Conversion of C:G to T:A Base Pairs in Zebrafish |
| title_fullStr | BE4max and AncBE4max Are Efficient in Germline Conversion of C:G to T:A Base Pairs in Zebrafish |
| title_full_unstemmed | BE4max and AncBE4max Are Efficient in Germline Conversion of C:G to T:A Base Pairs in Zebrafish |
| title_short | BE4max and AncBE4max Are Efficient in Germline Conversion of C:G to T:A Base Pairs in Zebrafish |
| title_sort | be4max and ancbe4max are efficient in germline conversion of c g to t a base pairs in zebrafish |
| topic | zebrafish base editing genome editing BE4max AncBE4max |
| url | https://www.mdpi.com/2073-4409/9/7/1690 |
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