CRISPR Synthetic Guide RNA Services

We continue to provide crRNAs and tracrRNAs services at GenScript. Email [email protected] to learn more or Get a Quote.

Why not trying our new synthetic sgRNA service, which eliminates the need for annealing the two RNA oligos, as well as having increased stability and editing efficiency?

CRISPR/Cas9 system can be delivered into cells in many forms, including plasmids, lentivirus, adeno-associated virus, and ribonucleoprotein (RNP) complexes. CRISPR/Cas9 ribonucleoprotein (RNP) system are composed of Cas9 protein and either a single guide RNA (sgRNA) or a CRISPR RNA (crRNA) : trans-activating crRNA (tracrRNA) duplex.

• The crRNAs are 20 nt RNA oligonucleotides designed to be complementary to a genomic target. crRNA sequence design utilizes standard gRNA design principles and sequences can be easily selected from the GenScript gRNA database or customized using the GenScript gRNA design tool.
• The tracrRNAs are 67 nt RNA oligonucleotides which together with the crRNA and Cas9 nuclease, form the activated CRISPR/Cas9 RNP complex.
• A sgRNA is a single RNA oligo that contains both the crRNA sequence, which can identify target genomic sequence, and the tracrRNA sequence, which can form complex with Cas9 protein.

When either a sgRNA or a crRNA : tracrRNA duplex forms complex with Cas9 protein, they can together, create double strand breaks at a locus complementary to the 20 nt guide RNA sequence, 3-4 bp upstream from a protospacer adjacent motif (PAM) sequence (5'-NGG-3').

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^1,2. 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.

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

CRISPR Handbook

CRISPR Ribonucleoprotein (RNP) User Manual

• How many sgRNA or crRNA sequences are needed for targeted knock-out? Read More »

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.

• What are the advantages of using synthetic sgRNA for gene editing compared to the traditional plasmids or virus based gRNA delivery methods? Read More »

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

• What is the different between using synthetic sgRNA vs using crRNA: tracrRNA duplex? Read More »

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.

• What is the advantage of your sgRNAs compared to sgRNA synthesized from in vitro transcription? Read More »

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. 

• Why should I choose modified sgRNA versus unmodified versions? Read More »

Our modified sgRNA has 2’-O-methyl and phosphorothioate modifications at the first three 5’ and 3’ terminal RNA residues.2′ O-Methyl oligo modification is best characterized as a RNA analog which offers stability against hydrolysis and nucleases. The phosphorothioate (PS) modification renders the internucleotide linkage more resistant to nuclease degradation.