CRISPR RNAs

GenCrispr/Cas9 Genome Editing Products

 

CRISPR/Cas9 technology is a powerful technique that has revolutionized the field of sequence-specific gene editing due to it's high precision, simplicity and flexibility. The CRISPR/Cas9 system consists of two major components: the Cas9 nuclease and the sequence-specific guide RNA (gRNA). They can form a very stable ribonucleoprotein (RNP) complex. Once a specific DNA region is matched by the gRNA carrying Cas9, the target DNA is cleaved with a double stranded break. Cas9 RNP complex can be introduced into cells directly and act immediately after it enters the cell without transcription and translation. By matching the gRNA sequence with different functional domains, the CRISPR/Cas9 system and its variants have been used for a broad range of gene editing applications (knock-out, knock-in, gene up/down, genome purification and visualization et. al.).

GenScript offers multiple kits to assemble your own Cas9 and gRNA constructs, as well as purified Cas9 enzymes for in vitro and in vivo use.


CRISPR/Cas9 Synthetic sgRNA and crRNA Service

CRISPR/Cas9 technology is a powerful tool for creating gene knock-ins and knock-outs. Using the ribonucleoprotein (RNP) system for delivering CRISPR/Cas9 has many advantages over the traditional plasmids or virus based delivery methods.


Why Choose CRISPR/Cas9 Ribonucleoprotein (RNP) System


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 gRNAdatabase 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').



Studies show that sgRNA has better stability than crRNA:tracrRNA when duplexed with Cas9.


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 efficiency1,2. 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/Cas9 crRNAs and tracrRNAs


CRISPR/Cas9 sgRNA


·         ISO9001 certificated, unmatchable quality assurance

·         Offer modified sgRNAs for better stability and editing efficiency

·         RNase-free HPLC guaranteeing more than 90% purity

·         97-103nt length range, allowing for greater flexibility in design

·         ESI-MS guaranteeing mass variation less than 1‰

·         4 nmol guaranteed delivery quantity


Product

Length

Quantity

GenCRISPR sgRNA

97 to 103 nt

4 nmol

Modified GenCRISPR sgRNA (2'-O-methyl and phosphorothioate modifications at the first three 5' and 3' terminal RNA residues)

97 to 103 nt

4 nmol


CRISPR/Cas9 tracrRNAs


GenScript CRISPR/Cas9 trans-activating crRNAs (tracrRNAs) are 67 nt RNA oligonucleotides which together with the crRNA and Cas9 nuclease, form the activated CRISPR/Cas9 ribonucleoprotein complex.


Product

Quantity

GenCRISPR tracrRNA

5 nmol

GenCRISPR tracrRNA

10 nmol

GenCRISPR tracrRNA

20 nmol

 

Nuclease-Free Annealing Buffer


Product

Concentration

Quantity

Nuclease-Free Annealing Buffer

5X

100 µL

Nuclease-Free Annealing Buffer

5X

1 mL



HPRT crRNA Positive Control and Primer Mix


Human HPRT (hypoxanthine phosphoribosyltransferase) is a housekeeping gene commonly used as positive control for testing CRISPR/Cas9 cleavage efficiency.


GenScript offers:


·         Pre-validated human HPRT crRNA, which after duplex with tracrRNA, can be used as positive controls for your CRISPR experiments. 

·         Human HPRT primer mix for assessing CRISPR cleavage efficiency via PCR.


Product

Quantity

Human HPRT crRNA

2 nmol

Human HPRT Primer Mix

2 nmol


CRISPR/Cas9 Control Kit


GenScript offers pre-duplexed human HPRT crRNA:tracrRNA as positive controls for your CRISPR experiments. HPRT (hypoxanthine phosphoribosyltransferase) is a housekeeping gene and commonly used control has already been pre-validated for efficient CRISPR cleavage. The kit comes with an HPRT primer mix for PCR analysis.


Product

GenCRISPR/Cas9 Control Kit


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