Site-specific randomization of the endogenous genome by a regulatable CRISPR-Cas9 piggyBac system in human cells
Randomized mutagenesis at an endogenous chromosomal locus is a promising approach for protein engineering, functional assessment of regulatory elements, and modeling genetic variations. In mammalian cells, however, it is challenging to perform site-specific single-nucleotide substitution with single...
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| Veröffentlicht in: | Scientific reports 2018-01, Vol.8 (1), p.310, Article 310 |
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| creator | Ishida, Kentaro Xu, Huaigeng Sasakawa, Noriko Lung, Mandy Siu Yu Kudryashev, Julia Alexandra Gee, Peter Hotta, Akitsu |
| description | Randomized mutagenesis at an endogenous chromosomal locus is a promising approach for protein engineering, functional assessment of regulatory elements, and modeling genetic variations. In mammalian cells, however, it is challenging to perform site-specific single-nucleotide substitution with single-stranded oligodeoxynucleotide (ssODN) donor templates due to insufficient homologous recombination and the infeasibility of positive selection. Here, we developed a DNA transposon based CRISPR-Cas9 regulated transcription and nuclear shuttling (CRONUS) system that enables the stable transduction of CRISPR-Cas9/sgRNA in broad cell types, but avoids undesired genome cleavage in the absence two chemical inducing molecules. Highly efficient single nucleotide alterations induced randomization of desired codons (up to 4 codons) at a defined genomic locus in various human cell lines, including human iPS cells. Thus, CRONUS provides a novel platform for modeling diseases and genetic variations. |
| doi_str_mv | 10.1038/s41598-017-18568-4 |
| format | Article |
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In mammalian cells, however, it is challenging to perform site-specific single-nucleotide substitution with single-stranded oligodeoxynucleotide (ssODN) donor templates due to insufficient homologous recombination and the infeasibility of positive selection. Here, we developed a DNA transposon based CRISPR-Cas9 regulated transcription and nuclear shuttling (CRONUS) system that enables the stable transduction of CRISPR-Cas9/sgRNA in broad cell types, but avoids undesired genome cleavage in the absence two chemical inducing molecules. Highly efficient single nucleotide alterations induced randomization of desired codons (up to 4 codons) at a defined genomic locus in various human cell lines, including human iPS cells. 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In mammalian cells, however, it is challenging to perform site-specific single-nucleotide substitution with single-stranded oligodeoxynucleotide (ssODN) donor templates due to insufficient homologous recombination and the infeasibility of positive selection. Here, we developed a DNA transposon based CRISPR-Cas9 regulated transcription and nuclear shuttling (CRONUS) system that enables the stable transduction of CRISPR-Cas9/sgRNA in broad cell types, but avoids undesired genome cleavage in the absence two chemical inducing molecules. Highly efficient single nucleotide alterations induced randomization of desired codons (up to 4 codons) at a defined genomic locus in various human cell lines, including human iPS cells. 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In mammalian cells, however, it is challenging to perform site-specific single-nucleotide substitution with single-stranded oligodeoxynucleotide (ssODN) donor templates due to insufficient homologous recombination and the infeasibility of positive selection. Here, we developed a DNA transposon based CRISPR-Cas9 regulated transcription and nuclear shuttling (CRONUS) system that enables the stable transduction of CRISPR-Cas9/sgRNA in broad cell types, but avoids undesired genome cleavage in the absence two chemical inducing molecules. Highly efficient single nucleotide alterations induced randomization of desired codons (up to 4 codons) at a defined genomic locus in various human cell lines, including human iPS cells. 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| subjects | 45/100 45/41 45/44 631/1647/1513/1967/3196 631/532/2064/2158 631/61/2300/1514 Cell lines Cells, Cultured Codon - genetics Codons CRISPR CRISPR-Cas Systems Deoxyribonucleic acid DNA DNA Transposable Elements Female Gene Editing - methods Genetic diversity Genomes HEK293 Cells Homologous recombination Humanities and Social Sciences Humans Male Mammalian cells multidisciplinary Mutagenesis Mutagenesis, Site-Directed - methods Positive selection Protein engineering Random Allocation Regulatory sequences RNA, Guide, CRISPR-Cas Systems - genetics Science Science (multidisciplinary) Transcription Transduction, Genetic - methods |
| title | Site-specific randomization of the endogenous genome by a regulatable CRISPR-Cas9 piggyBac system in human cells |
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