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News & Blogs » CRISPR News » In Vitro-Transcribed CRISPR sgRNAs Induce Innate Immune Responses in Human Cells
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In Vitro-Transcribed CRISPR sgRNAs Induce Innate Immune Responses in Human Cells

crispr, genome editing, kidney transplant, organ transplant, hepatitis C, hepatitis C virus, HCVCRISPR/Cas9-mediated gene editing is a powerful technique that allows you to create knock-in/out mutations in any gene and any cell. CRISPR/Cas9 system can be delivered into cells in many forms, including plasmids, lentivirus, adeno-associated virus, and ribonucleoprotein (RNP) complexes. The RNP system, consisting of Cas9 proteins and single-guide RNAs (sgRNA), are gaining more popularity due many advantages it has over the other methods.

The advantages of using the RNP system include:

  • DNA free
  • Detectable at high levels shortly after transfection
  • Quickly cleared from the cell for less off-target effects
  • Highly efficient even in hard-to-transfect cells
  • Best for in vivo studies
  • In particular, studies show that sgRNA has better stability than crRNA:tracrRNA when duplexed with Cas9.

sgRNAs are traditionally produced through in vitro transcription using phage RNA polymerase. These in vitro transcribed sgRNAs contain a 5’-triphosphate, which was thought to trigger immune response in many cell types. Kim et al. recently published their study in Genome Research, which showed that sgRNAs with 5’-triphosphate modifications produced through in vitro-transcription can indeed induce innate immune responses and lead to cytotoxicity in human and murine cells. However, chemically synthesized sgRNAs without the 5’-triphosphate modifications demonstrated much better editing efficiency in cells, thus supporting that chemically synthesized sgRNAs are the most ideal reagent for CRISPR genome editing up till now.


Kim et al.CRISPR RNAs trigger innate immune responses in human cells. Genome Res. 2018. 28: 367-373.
DOI: 10.1101/gr.231936.117

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