Top Questions

Can gBlocks® Gene Fragments be used for microinjection as single guide RNA (sgRNA) expression cassettes for CRISPR applications?

A recent report compared the efficiency of microinjection of DNA versus RNA in mouse embryos [1]. While it was shown that DNA is effective, in vitro transcribed RNA was observed to be more efficient.

Typically, microinjections for CRISPR applications are performed using in vitro transcribed Cas9 and sgRNA rather than native dsDNA. gBlocks® Gene Fragments are ideal for use as template for in vitro transcription and will work well in these applications [2,3]. However, long RNAs are known to trigger the innate immune response in many cells, which increases the expression of dozens of genes and can affect cell viability and general health [4–7].

References

  1. Horii T, Arai Y, et al. (2014) Validation of microinjection methods for generating knockout mice by CRISPR/Cas-mediated genome engineering. Sci Rep, 4:4513.
  2. Niu Y, Shen B, et al. (2014) Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos. Cell, 156(4):836–843.
  3. Wang H, Yang H, et al. (2013) One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell, 153(4):910–918.
  4. Gantier MP and Williams BR (2007) The response of mammalian cells to double-stranded RNA. Cytokine Growth Factor Rev, 18:363–371.
  5. Judge A and MacLachlan I (2008) Overcoming the innate immune response to small interfering RNA. Hum Gene Ther, 19:111–124.
  6. Hornung V, Hartmann R, et al. (2014) OAS proteins and cGAS: unifying concepts in sensing and responding to cytosolic nucleic acids. Nat Rev Immunol, 14:521–528.
  7. Robbins M, Judge A, MacLachlan I (2009) siRNA and innate immunity. Oligonucleotides, 19:89–102.
Tags:
  • gBlocks
  • CRISPR
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