| [PMID]: | 28666380 |
| [Au] Autor: | Luo W; Galvan DL; Woodard LE; Dorset D; Levy S; Wilson MH |
| [Ad] Endereço: | Department of Veterans Affairs, Nashville, TN 37212 USA and Department of Medicine, Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232, USA. |
| [Ti] Título: | Comparative analysis of chimeric ZFP-, TALE- and Cas9-piggyBac transposases for integration into a single locus in human cells. |
| [So] Source: | Nucleic Acids Res;45(14):8411-8422, 2017 Aug 21. |
| [Is] ISSN: | 1362-4962 |
| [Cp] País de publicação: | England |
| [La] Idioma: | eng |
| [Ab] Resumo: | Integrating DNA delivery systems hold promise for many applications including treatment of diseases; however, targeted integration is needed for improved safety. The piggyBac (PB) transposon system is a highly active non-viral gene delivery system capable of integrating defined DNA segments into host chromosomes without requiring homologous recombination. We systematically compared four different engineered zinc finger proteins (ZFP), four transcription activator-like effector proteins (TALE), CRISPR associated protein 9 (SpCas9) and the catalytically inactive dSpCas9 protein fused to the amino-terminus of the transposase enzyme designed to target the hypoxanthine phosphoribosyltransferase (HPRT) gene located on human chromosome X. Chimeric transposases were evaluated for expression, transposition activity, chromatin immunoprecipitation at the target loci, and targeted knockout of the HPRT gene in human cells. One ZFP-PB and one TALE-PB chimera demonstrated notable HPRT gene targeting. In contrast, Cas9/dCas9-PB chimeras did not result in gene targeting. Instead, the HPRT locus appeared to be protected from transposon integration. Supplied separately, PB permitted highly efficient isolation of Cas9-mediated knockout of HPRT, with zero transposon integrations in HPRT by deep sequencing. In summary, these tools may allow isolation of 'targeted-only' cells, be utilized to protect a genomic locus from transposon integration, and enrich for Cas9-mutated cells. |
| [Mh] Termos MeSH primário: |
Técnicas de Inativação de Genes/métodos
Marcação de Genes/métodos
Técnicas de Transferência de Genes
Mutagênese Insercional/métodos
|
| [Mh] Termos MeSH secundário: |
Proteínas de Bactérias/genética
Sistemas CRISPR-Cas/genética
Linhagem Celular Tumoral
Elementos de DNA Transponíveis/genética
Endonucleases/genética
Seres Humanos
Hipoxantina Fosforribosiltransferase/genética
Hipoxantina Fosforribosiltransferase/metabolismo
Proteínas Recombinantes de Fusão/genética
Reprodutibilidade dos Testes
Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética
Efetores Semelhantes a Ativadores de Transcrição/genética
Transposases/genética
Dedos de Zinco/genética
|
| [Pt] Tipo de publicação: | COMPARATIVE STUDY; JOURNAL ARTICLE |
[Nm] Nome de substância:
| 0 (Bacterial Proteins); 0 (DNA Transposable Elements); 0 (Recombinant Fusion Proteins); 0 (Transcription Activator-Like Effectors); EC 2.4.2.8 (Hypoxanthine Phosphoribosyltransferase); EC 2.7.7.- (Transposases); EC 3.1.- (Cas9 endonuclease Streptococcus pyogenes); EC 3.1.- (Endonucleases); EC 3.1.- (Transcription Activator-Like Effector Nucleases) |
| [Em] Mês de entrada: | 1710 |
| [Cu] Atualização por classe: | 171023 |
[Lr] Data última revisão:
| 171023 |
| [Sb] Subgrupo de revista: | IM |
| [Da] Data de entrada para processamento: | 170702 |
| [St] Status: | MEDLINE |
| [do] DOI: | 10.1093/nar/gkx572 |
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