Is Targeted Insertion of Short DNA Fragments into Mammalian Cells Possible?


Recently the Targeted Insertion of Short DNA Fragments into Mammalian Cells was Shown to be Possible with the help of CRISPR Cas!

The direct injection of Cas9 mRNA, sgRNAs, and DNA oligos or vectors into mammalian cells was reported as being possible. Yang et al. in 2013 demonstrated that CRISPR Cas technology can be used for efficient one-step insertions of a short epitope or longer fluorescent tags into precise genomic locations. Designed oligonucleotides containing the 34 bp loxP site and a 6 bp EcoRI site flanked by 60 bps sequences on each side adjoining the DSBs were used in this study. The study showed that this approach facilitates the generation of mice carrying reporters in endogenous genes. The researchers used mice and embryos carrying reporter constructs in the Sox2, the Nanog and the Oct4 gene that were derived from zygotes injected with Cas9 mRNA, sgRNAs, and DNA oligos or vectors encoding a tag or a fluorescent marker. Furthermore, the microinjection of two Mecp2-specific sgRNAs, Cas9 mRNA, and two different oligos encoding loxP sites into fertilized eggs, allowed the one-step generation of conditional mutant mice. In addition, the researchers showed that the introduction of two spaced sgRNAs targeting the Mecp2 gene produced mice carrying defined deletions of about 700 bp. The use of Southern analyses allowed the scientist to show that mosaicism occurred in 17% (1/6) to 40% (20/49) of the targeted mice and ES cell lines. This indicates that the insertion of the transgenes had occurred after the zygote stage.

Mashiko et al. in 2013 also reported that CRISPR Cas mediated genome editing has been successfully demonstrated in mammalian cells, and reported a further application for generating mutant mice by injecting humanized Cas9 (hCas) mRNA and single guide RNA into fertilized eggs. Circular plasmids expressing hCas9 and sgRNA were injected into mouse zygotes and mutant mice were obtained within a month in this study. The researchers report that the pronuclear injection of circular plasmid expressing hCas9/sgRNA complex is a rapid, simple, and reproducible method for targeted mutagenesis.

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Reference

Daisuke Mashiko, Yoshitaka Fujihara, Yuhkoh Satouh, Haruhiko Miyata, Ayako Isotani & Masahito Ikawa; Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA. Scientific Reports (2013) 3, Article number: 3355 doi:10.1038/srep03355.

Hui Yang,Haoyi Wang,Chikdu S. Shivalila,Albert W. Cheng,Linyu Shi,and Rudolf Jaenisch; One-Step Generation of Mice Carrying Reporter and Conditional Alleles by CRISPR/Cas-Mediated Genome Engineering. Cell 154, 1370–1379, September 12, 2013.



Categories: Artificial Nucleic Acids, Bioinformatics, Cellular Reprogramming, CRISPR Cas, DNA, DNA Structure, DNA Synthesis, Epigenetics, Gene Editing, Gene Expression, Genetics, Genome, In Vitro Transcription, lncRNA, Long noncoding RNA, Molecular Probes, non-coding RNAs, Oligonucleotide Synthesis, Protein, Protein Families, Protein Structure, Regulatory RNA, RNA, RNA Editing, RNA FISH, RNA Structure, RNA Synthesis, RNA World, Synthesis

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1 reply

  1. Insightful and incisive summary of CRISPR genome engineering!

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