GenCRISPR Synthetic sgRNA

Unlike the traditional plasmids or lentivirus delivery methods, CRISPR/Cas9 RNP are delivered as intact complexes, and do not require cellular expression, thus has many advantages.

• 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

Recently, several studies have showed that sgRNA has better stability than crRNA:tracrRNA when duplexed with Cas9, thus leading to higher editing efficiency. Synthesis of 100 nt long sgRNAs was traditionally possible through in vitro-transcription (IVT) using phage RNA polymerase. These in vitro transcribed sgRNAs contain a 5’-triphosphate, which was thought to trigger immune response in many cell types. A recent study 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 now¹.

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

A minimum of 3 crRNA sequences are recommended to ensure knock-out and experimental accuracy. Independently obtained knock-out mutants provide redundancy to safeguard against any hidden off-target effects.

Unlike the traditional plasmids or lentivirus delivery methods, CRISPR/Cas9 RNP are delivered as intact complexes, and do not require cellular expression, thus has many advantages.

• 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

When using sgRNA, there is no need for annealing crRNA and tracrRNA duplex prior to use. More importantly, several studies have showed that sgRNA has better stability than crRNA:tracrRNA when duplexed with Cas9, thus leading to higher editing efficiency^1,2.

1. Hendel, et al., Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nat. Biotechnol., 33 (2015) 985-989.
2. Ryan et al., Improving CRISPR–Cas specificity with chemical modifications in single-guide RNAs. Nucleic Acids Research, 46 (2018) 2: 792–803.

Synthesis of 100 nt long sgRNAs was traditionally possible through in vitro-transcription (IVT) using phage RNA polymerase. These in vitro transcribed sgRNAs contain a 5’-triphosphate, which was thought to trigger immune response in many cell types. A recent study 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 now³.

3. Kim et al. CRISPR RNAs trigger innate immune responses in human cells. Genome Res. 2018. 28: 367-373.