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.
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