Identifying optimal conditions for precise knock-in of exogenous DNA into the zebrafish genome.
| Title: | Identifying optimal conditions for precise knock-in of exogenous DNA into the zebrafish genome. |
|---|---|
| Authors: | Oikemus S; Department of Molecular, Cell, and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA.; Hu K; Department of Molecular, Cell, and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA.; Shin M; Department of Molecular, Cell, and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA.; Idrizi F; Department of Molecular, Cell, and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA.; Goodman-Khan A; Department of Molecular, Cell, and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA.; Kolb A; Department of Molecular, Cell, and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA.; Ghanta KS; RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA.; Lee J; RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA.; Wagh A; RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA.; Wolfe SA; Department of Molecular, Cell, and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA.; Zhu LJ; Department of Molecular, Cell, and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA.; Watts JK; RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA.; Lawson ND; Department of Molecular, Cell, and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA. |
| Source: | Development (Cambridge, England) [Development] 2025 Jun 15; Vol. 152 (12). Date of Electronic Publication: 2025 Jun 19. |
| Publication Type: | Journal Article |
| Language: | English |
| Journal Info: | Publisher: Company Of Biologists Limited Country of Publication: England NLM ID: 8701744 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1477-9129 (Electronic) Linking ISSN: 09501991 NLM ISO Abbreviation: Development Subsets: MEDLINE |
| Imprint Name(s): | Publication: Cambridge Eng : Company Of Biologists Limited; Original Publication: [Cambridge] : Company of Biologists, [c1987- |
| MeSH Terms: | Zebrafish*/genetics ; Genome*/genetics ; Gene Editing*/methods ; Gene Knock-In Techniques*/methods ; DNA*/genetics; CRISPR-Cas Systems/genetics ; Recombinational DNA Repair/genetics ; Animals ; DNA Breaks, Double-Stranded |
| Abstract: | CRISPR nucleases can be used to insert exogenous DNA into the zebrafish genome by homology-dependent repair (HDR), although germline transmission rates for precise edits remain quite low. Comparative studies to optimize HDR parameters for introducing base pair changes using short-read deep sequencing have been successful, but similar analysis for insertions is challenging due to read-length constraints. Here, we quantified editing outcomes using long-read sequencing to identify optimal template and CRISPR parameters for precise targeted insertion in zebrafish. Through side-by-side comparisons, we found that chemically modified templates out-perform those released in vivo from a plasmid, while Cas9 and Cas12a nucleases performed similarly for targeted insertion. Consistent with previous studies, precise editing rates were dependent on the distance between a double-strand break and the inserted sequence. We further found that non-homologous base pairs in homology templates significantly reduced precise editing rates. Using optimized parameters, we consistently achieved germline founder rates of greater than 20% for precise insertions across four loci. Together, our quantitative analyses identified optimal conditions for precise insertion of exogenous DNA into the zebrafish genome.; (© 2025. Published by The Company of Biologists.) |
| Competing Interests: | Competing interests The authors declare no competing or financial interests. |
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| Grant Information: | R35HL171534 United States NH NIH HHS; University of Massachusetts Medical School; R35HL171534 United States HL NHLBI NIH HHS; 1UG3TR002668 United States NH NIH HHS; R35 HL171534 United States HL NHLBI NIH HHS; R21OD030004 United States NH NIH HHS; UG3 TR002668 United States TR NCATS NIH HHS; R21 OD030004 United States OD NIH HHS; R21OD030004 NIH Office of the Director |
| Contributed Indexing: | Keywords: CRISPR; Genome editing; Homology-dependent repair; Zebrafish |
| Substance Nomenclature: | 9007-49-2 (DNA) |
| Entry Date(s): | Date Created: 20250530 Date Completed: 20250619 Latest Revision: 20250704 |
| Update Code: | 20260130 |
| PubMed Central ID: | PMC12211562 |
| DOI: | 10.1242/dev.204571 |
| PMID: | 40446205 |
| Database: | MEDLINE |
Journal Article