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Nucleic Acids Res. 2013 Aug;41(14):e141. doi: 10.1093/nar/gkt464. Epub 2013 Jun 6.

Chromosomal deletions and inversions mediated by TALENs and CRISPR/Cas in zebrafish.

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1
Key Laboratory of Cell Proliferation and Differentiation of Ministry of Education, College of Life Sciences, Peking University, Beijing 100871, China.

Abstract

Customized TALENs and Cas9/gRNAs have been used for targeted mutagenesis in zebrafish to induce indels into protein-coding genes. However, indels are usually not sufficient to disrupt the function of non-coding genes, gene clusters or regulatory sequences, whereas large genomic deletions or inversions are more desirable for this purpose. By injecting two pairs of TALEN mRNAs or two gRNAs together with Cas9 mRNA targeting distal DNA sites of the same chromosome, we obtained predictable genomic deletions or inversions with sizes ranging from several hundred bases to nearly 1 Mb. We have successfully achieved this type of modifications for 11 chromosomal loci by TALENs and 2 by Cas9/gRNAs with different combinations of gRNA pairs, including clusters of miRNA and protein-coding genes. Seven of eight TALEN-targeted lines transmitted the deletions and one transmitted the inversion through germ line. Our findings indicate that both TALENs and Cas9/gRNAs can be used as an efficient tool to engineer genomes to achieve large deletions or inversions, including fragments covering multiple genes and non-coding sequences. To facilitate the analyses and application of existing ZFN, TALEN and CRISPR/Cas data, we have updated our EENdb database to provide a chromosomal view of all reported engineered endonucleases targeting human and zebrafish genomes.

PMID:
23748566
PMCID:
PMC3737551
DOI:
10.1093/nar/gkt464
[Indexed for MEDLINE]
Free PMC Article
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