CRISPR/Cas9 sgRNA Synthesis

CRISPR/Cas9 sgRNA Synthesis

High-Purity sgRNA, Optimized for Precise CRISPR/Cas9 Editing

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Overview

Our high-purity, 100% chemically synthesized CRISPR/Cas9 sgRNA deliver unparalleled efficiency, specificity, and consistency, enabling reliable gene editing in research and therapeutic development.

Pricing

97-103 nt

2 nmol

$79

$119

4 nmol

$149

$209

10 nmol

$299

$459

50 nmol

$839

$1299

100 nmol

$1899

GenScript offers INDEdit and cGMP-grade products for IND submissions and clinical research. Learn more.

Need help from an expert?

Need help with your sgRNA design?

Access our intelligent design tools below to design and order Cas9 sgRNA (and DNA templates, if needed) for your CRISPR/Cas knockout or HDR knock-in experiments:

Already have your sgRNA design? Use our simple order form to order or get a quote:

Cas9 sgRNA

Prime Editing gRNA

Base Editing gRNA

Cas12a crRNA

cGMP gRNA

GenExact ssDNA

GenWand dsDNA

GenCircle dsDNA

cGMP ssDNA/dsDNA

Non-viral CAR-T Knock-in Optimization Kits

sgRNA Design Tool

HDR Design Tool

pegRNA Design Tool

cGMP Cas Proteins

gRNA List

gRNA Libraries

Cell Engineering

Microbial Editing

Cas9 Nucleases

Why GenScript?

50,000+

We deliver gRNAs/year

4

Production in as few as business days

100%

We deliver On-time

Advantages

EasyEdit

Synthetic EasyEdit sgRNAs now starting at only $79/2nmol!

✔ Option to deliver in 96-well plates supporting high throughput screening

Most economical solution

High editing efficiency

Flexible at scale

EasyEditThe easy yet effective one-step solution Starting at $79/2nmol!

HPLC purified sgRNAs minimize off-targeting and cytotoxicity

✔ >90% purity guaranteed

Minimal impact for cell viability

Reduced off-targeting from truncated guides

Ideal for primary cells & stem cells

cGMP/GMP-likeSupporting IND filing and clinical trials

GenScript now offers full cGMP and INDEdit sgRNA

✔ Supporting IND filing and clinical trials

State-of-the-art production facility

Clean suite with class A isolator in a class C background

Comprehensive QA/QC

Proprietary NGS method to verify sgRNA identity

Trusted by scientific partners globally

Successfully delivered 130+ cGMP batches

Science Never Stops – Neither Do We!

Support seamless transition every step of the way

With a robust global infrastructure, financial resilience, and years of CRISPR expertise, we ensure your science never skips a beat. Ready for an Upgrade? Our dedicated Transition Support Team is here to assist every step of the way!

Download our Step-By-Step Switching Support Guide to learn how we enable seamless transitions

Download our Switching Checklist to ensure no critical steps are missed

Book your free consultation to explore a customized transition plan

Our dedicated Transition Support Team is ready to help

Service Details

Length

Default Modifications

2’-O-methyl and phosphorothioate at 3’ and 5’ ends

Purification

Desalt

HPLC

Quantity

2-100 nmol

QC Reports

COA and MS reports

COA, MS, and HPLC reports

Delivery Time

Starting from 7 BDs

Starting from 9 BDs

Catalog Products

KIOK3

Ultra eSpCas9

$379.00 200 μg

KIOK4

Ultra WTSpCas9

$249.00 200 μg

RC00001

GenCRISPR™ NLS-wtSpCas9, his-tag

$150.00 0.1 mg 1 mg 3 mg

RP-A00018

eSpCas9 mRNA(Cap1, m1Ψ)

$350.00 0.2 mg 1 mg

RP-A00019

GenLNP-A01-eSpCas9 mRNA(m1Ψ)-TRAC sgRNA

$600.00 0.05 mg

RP-A00020

GenLNP-S01-eSpCas9 mRNA(m1Ψ)-TRAC sgRNA

RP-A00047

saCas9 mRNA (Cap1, m1Ψ)

RP-A00050

eSpCas9 mRNA(Cap 1,5-MOU)

Z03621

GenCRISPR™ Ultra NLS-Cas9-Research

$695.00 0.5 mg 1 mg 5 mg

Z03622

GenCRISPR™ Ultra eSpCas9-2NLS-Research

$765.00 0.5 mg 1 mg 5 mg

Looking for guides for other editing systems?

97-140 nt, enabling both conventional + transformer base editing

Up to 266 nt- the longest from any vendor- with editing efficiency of over 60%

40 to 49 nt, with superior quality and editing efficiency up to 98%

GenScript can also synthesize desalt and HPLC purified guide RNA compatible with saCas9, Cas12f/CasMINI, Cas13, and other Cas nuclease variants.

Click here to enter your guide sequence (target sequence + scaffold sequence) and order online.

Testimonials

Dr. Yoel Shiboleth

TargetGene Biotechnologies LTD

"TargetGene is a long-time, highly satisfied customer of GenScript and we have been purchasing all of our custom gRNAs for the T-GEE platform from GenScript […] Our decisive choice of GenScript arises from the high quality of the products, full flexibility in guide scaffold sequence, excellent web-interface, competitive pricing at different production scales, very quick and professional response of both the Singapore and local representatives, and rapid delivery times. We highly recommend GenScript to our peers!"

Ilknur Sur Erdem

University of Oxford

"With the gRNA from GenScript, we had a successful knockout. Thank you for very good service."

Dr. Xinlai Cheng

Buchmann Institute for Molecular Life Sciences, Goethe University Frankfurt

Case Studies

Reliable and Consistent Editing Results

GenScript EasyEdit sgRNA achieved >80% KO efficiency for all three gene targets tested, comparable to other vendors. sgRNAs were designed for 3 randomly selected gene targets (Seq_1, Seq_2, Seq_3), and KO efficiency of sgRNAs from different vendors were examined in Jurkat cells.

Superior purity compared to competitor products

GenScript EasyEdit sgRNA demonstrates higher purity compared to competitor products. UPLC analysis of sgRNA purity from different vendors for sgRNAs designed for 3 randomly selected gene targets (Seq_1, Seq_2, Seq_3).

Higher % of full-length sgRNA sequences compared to competitor products

GenScript EasyEdit sgRNA exhibited minimal non-specific peaks in MS analysis (a) and a higher percentage of full-length sgRNA sequences in NGS analysis (b) compared to competitor products for the same sgRNA sequence design.

SafeEdit sgRNA eliminates off-targeting from truncated oligos

High purity SafeEdit sgRNA ensures minimal impact for cell viability

SafeEdit sgRNA exhibits high editing efficiency across a variety of loci and cell types

Editing efficiencies up to 97% were achieved

Resources

RNP user manual

A guide on how to use CRISPR RNP for targeted genome editing.

CRISPR Knock-in Comprehensive Guide

Step-by-step instructions for your CRISPR knock-in experiments.

cGMP sgRNA: A Strategic Approach to Sample Impurity Identification and Evaluation for Optimized Gene Editing in Gene and Cell Therapy

GenCRISPR gRNA & HDR Template Design Tools

For knockout and knock-in designs, with off-target analysis.

FAQs

Technical questions

Design tool questions

Experiment questions

A minimum of three (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 plasmid or lentiviral delivery methods, CRISPR/Cas9 ribonucleoprotein (RNP) are delivered as intact complexes and do not require cellular expression. This method thus has many advantages:

Faster manufacturing: Synthetic sgRNA can be made in as little as three (3) business days using chemical synthesis and arrives ready to use- compared to a week or more of lab work required to produce gRNA via plasmid or lentiviral delivery.

DNA-free: Avoiding the delivery of foreign DNA into the cell eliminates any opportunity for transgene integration into the cell genome.

Detectable at high levels shortly after transfection: Editing activities can take place more quickly, as synthetic sgRNA doesn't need to be transcribed in the cell.

Quickly cleared from the cell for less off-target effects: The Cas-sgRNA RNP delivery vehicle is expressed transiently and degrades quickly, limiting the potential for any off-target editing.

Highly efficient even in hard-to-transfect cells: Experimental results have demonstrated high editing efficiency using synthetic sgRNA, regardless of the cell type used.

High packaging capacity: Our synthetic sgRNA can accommodate a length of up to 200nt.

Low immunogenicity (ideal for in vivo studies): Synthetic sgRNA consistently demonstrates lower immunogenicity and toxicity in edited cells compared to gRNA produced via plasmid or lentiviral delivery.

When using sgRNA, there is no need for a crRNA:tracrRNA annealing step 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 (such as GenCRISPR Synthetic sgRNAs) demonstrated much better editing efficiency in cells, thus supporting that chemically synthesized sgRNAs are the most ideal reagent for CRISPR genome editing currently available.3

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

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 an RNA analog which offers stability against hydrolysis and nucleases. The phosphorothioate (PS) modification renders the internucleotide linkage more resistant to nuclease degradation.

Our sgRNA can be delivered in either single tubes or 96-well plates, formatted as dry powder or suspended in TE buffer (pH 8.0, 100 µM) or nuclease-free water (100 µM).

We can chemically synthesize sgRNA up to 266 nt in length.

We can deliver chemically synthesized sgRNAs in an arrayed format, which is delivered in a 96-well plate. Each well contains custom-designed sgRNA targeting one gene of interest.

We offer sgRNA in grades ranging from RUO to cGMP:

EasyEdit sgRNA is purified by an optimized desalt method. Fast delivery and competitive pricing makes it ideal for screening or early stages of research.

SafeEdit sgRNA is purified by HPLC, resulting in a guaranteed minimum of 90% purity. This option is ideal for the validation stage of development.

For more information on our cGMP sgRNA, visit this page.

Designing a good sgRNA for your target depends on several factors, such as the specificity, efficiency, and compatibility of the sgRNA with your CRISPR system. Here are some general guidelines for sgRNA design:

Choose a sgRNA sequence that is complementary to your target DNA sequence and contains a protospacer adjacent motif (PAM) that matches the Cas protein you are using.

Avoid sgRNA sequences that have high similarity with non-target DNA sequences, as this may cause off-target effects. A good sgRNA has a high on-target score and low off-target score.

Optimize the GC content and length of the sgRNA sequence to improve the stability and activity of the sgRNA. A general rule of thumb is to have between 40% and 80% GC content and 17 to 24 nucleotides of spacer RNA.

A Doench Rule Set score is used to measure on-target efficiency; the higher the score, the higher the efficiency. Off-target scores are calculated based on the number of potential off-target binding sites within three (3) base mismatches. A lower score denotes lower off-target potential.

Yes! We can design gRNA for ANY gene editing system.

Currently, our online design tools support gRNA designs for Cas9, Cas12a, and prime editing. If you need to design gRNA for base editing or another gene editing system, please reach out a CRISPR expert at [email protected].

Currently, our design tools support editing in the following species:

For Cas9 and Cas12a editing: 14 species including human, mouse, rat, fruit fly, and Chinese hamster.

For prime editing: 8 species including human, mouse, and fruit fly.

If you need to design gRNA for another species, please reach out to a CRISPR expert at [email protected].

Yes, our HDR knock-in design tool can be used to design HDR templates and compatible sgRNAs for KI experiments.

The process flow diagram (Select Gene->Mutation-> Design Result) is located on the top right side of the design tool webpage. Click the corresponding step in the diagram to go back and review or edit previously entered information.

The 'primer design' option in our sgRNA design tool helps you design primers for sequencing. You can customize the approximate distance between sequencing primers and the melting temperature (Tm) range. The custom-designed primer sets are used for sequencing the target DNA sequence to verify if the desired genomic modification is created correctly after performing the edits.

If there are no suitable sgRNA cleavage sites within 30 bp of your targeted cleavage site, you may have to consider alternative strategies for gene editing. Some possible options are:

Use a different Cas variant that recognizes a different PAM sequence. For example, SaCas9 recognizes the 3’NNGRRT-5’ PAM site more frequently than the 3’NGG-5’ PAM site recognized by SpCas9.

Use a nickase variant of Cas9 to create single-strand breaks instead of double-strand breaks. Using two nickases with different sgRNAs, you can create staggered nicks on opposite strands of the target DNA, which HDR or NHEJ can repair.

Use a CRISPR-Cas12a system, which can generate longer overhangs at the cleavage site and increase the efficiency of HDR.

If you need assistance choosing an alternative strategy, feel free to contact one of our CRISPR experts at [email protected].

Our gRNA and HDR knock-in design tools accept gene symbols and gene IDs approved by HGNC. If you cannot find your target gene, please search the gene database for the correct gene name and ID. If you still can’t find your gene, please contact us for technical support at [email protected].

Yes, you can easily order sgRNA using your own sequence. Simply add your sequence to our sgRNA Ordering form here to get started.

To correctly add your crRNA protospacer sequence, follow these steps:

Identify the target DNA sequence that you want to edit using CRISPR-Cas9. The target sequence should be 20 nucleotides long, followed by a PAM sequence recognized by the Cas9 protein of your choice.

Design your crRNA protospacer sequence by copying the 20 nucleotides upstream of the PAM sequence, in the forward orientation. Do not include the PAM sequence in your crRNA protospacer sequence. For example, if your target DNA sequence is 5’-ATCGGACTAGCTAGCTAGCTAGG-3’, your crRNA protospacer sequence should be 5’-ATCGGACTAGCTAGCTAGCT-3’2.

Order your crRNA protospacer sequence. An online gRNA design tool can be used to design and evaluate your crRNA protospacer sequence. Ensure you verify the correct strand orientation and omit the PAM site.

Combine your crRNA protospacer sequence with a tracrRNA sequence that can hybridize with the crRNA and form a guide RNA (gRNA) complex. Alternatively, you can use a single guide RNA (sgRNA) that contains both the crRNA and the tracrRNA sequences in one molecule.

No. You do not need to include the PAM site in your synthesized or expressed gRNA. However, you must ensure the presence of an intact PAM, corresponding to the Cas nuclease variant used for your editing project, in your target sequence adjacent to the gRNA target.

To choose two gRNAs to multiplex when using a Cas9 nickase, you need to consider the following factors:

The gRNAs should target opposite DNA strands and generate nicks close enough to induce a double-strand break (DSB) by the cellular DNA repair machinery.

The optimal distance between the two nicks depends on the type of Cas9 nickase and the target sequence. For SpCas9 nickase, the distance should be within 1–100 bp, with 5–20 bp being the most efficient range. The distance may vary for other Cas9 nickases, such as SaCas9 or AsCas12a.

The gRNAs should have high on-target activity and low off-target activity. You can use online CRISPR design tools to design and evaluate your gRNAs.

The gRNAs should have compatible PAM sequences for the Cas9 nickase of your choice. For SpCas9 nickase, the PAM sequence is NGG. For other Cas9 nickases, the PAM sequence may differ.

If a PAM sequence is absent in your target, you cannot use the Cas9 nuclease to cut the target DNA sequence. The PAM sequence is essential for Cas9 to recognize and bind to the target DNA sequence. Without the PAM sequence, Cas9 will not be able to cleave the target DNA sequence, even if it is complementary to the guide RNA.

However, there are some possible solutions to overcome this limitation. One solution is to use a different Cas9 nuclease that recognizes a different PAM sequence. For example, the Cas9 nuclease of Streptococcus pyogenes (SpCas9) recognizes the PAM sequence 5’-NGG-3’, but the Cas9 nuclease of Neisseria meningitidis (NmCas9) recognizes the PAM sequence 5’-NNNNGATT-3’. Therefore, if your target DNA sequence does not have a 5’-NGG-3’ PAM sequence, you may be able to use NmCas9 instead of SpCas9 to edit your target.

Another solution is to engineer a Cas9 nuclease that recognizes a different PAM sequence. For example, the Cas9 nuclease of Francisella novicida (FnCas9) recognizes the PAM sequence 5’-NGG-3’ but has been engineered to recognize 5’-YG-3’ (where Y is a pyrimidine).4 This expands the range of possible target sites for Cas9 editing. Similarly, the Cpf1 nuclease of Francisella novicida (FnCpf1) recognizes the PAM sequence 5’-TTTN-3’ or 5’-YTN-3’. Cpf1 is another CRISPR-associated nuclease that can be used for genome editing.

4. Hirano et al., Structure and Engineering of Francisella novicida Cas9. Cell, 164 (2016) 950–961

A genomic cleavage detection assay, such as our GenCrispr Mutation Detection Kit, is a fast, T7 endonuclease I-based method to quantify how well your genome editing protocol causes insertions and deletions (indels) in the genome of your cell line. It is the quickest way to validate and select the best CRISPR-Cas9 gRNA and nuclease for your genome editing experiments.

The presence of additional PAM sites does not necessarily trigger off-target cleavage. PAM sites, defined as NGG, are quite common within any genome. Cleavage specificity is determined by the juxtaposition of your specific target to an NGG site. If there is some redundancy in the region you are trying to edit, you may be at a higher risk of off-site cleavage. If this is a particular problem, you may wish to explore an alternate Cas protein that uses a larger, less common PAM site.

We have recommended protocols for performing CRISPR knockout experiments available for download in our free CRISPR Ribonucleoprotein (RNP) User Manual.

Dissolve the powdered sgRNA in TE buffer to a concentration of 100 pmol/µL, or other desired concentration. Divide into aliquots as needed and store at -20℃, avoiding repeated freeze-thaw cycles. For long-term storage, store at -80℃.

Yes, using a validated control of a standard target region that cuts DNA using CRISPR can help optimize workflow conditions to ensure the highest editing efficiency before conducting the actual experiment or running in parallel to the main CRISPR experiments. Positive editing controls, negative editing controls, and mock controls are just a few examples of CRISPR controls that can be used in different ways to help assess how well workflow conditions are optimized and the editing efficiency.

You can add controls after designing your sgRNA in our GenCRISPR sgRNA design tool or order them directly via our Quick Order form here.

In addition to your sgRNA, CRISPR-mediated gene editing requires a Cas protein that can recognize and cleave your target DNA sequence. The most commonly used Cas protein is SpCas9, which requires a NGG PAM sequence adjacent to the target site. However, there are other Cas variants that can recognize different PAM sequences or have increased specificity. You can either express the Cas protein from a plasmid or deliver it as a purified protein (ribonucleoprotein, or RNP) into the cells.

Additionally, a donor (HDR) template can be used to provide the desired sequence change for precise editing. This is optional, but recommended if you want to introduce specific mutations, insertions, or deletions into your target gene. The donor template can be a single-stranded or double-stranded DNA molecule that has homology arms flanking the target site. Alternatively, you can use a base editor or a prime editor that can directly install the desired base pair conversions without requiring a donor template.

The stability of the RNP complex may vary depending on the type of Cas protein, the guide RNA, and the storage conditions. However, some studies have reported that the RNP complex can be stable for up to one year at –20°C or –80°C, without requiring any RNase inhibitors. The RNP complex can also retain its nuclease activity after multiple freeze-thaw cycles. However, it is recommended to use fresh RNP complexes whenever possible and to avoid repeated freeze-thaw cycles, as they may affect the quality and efficiency of the RNP complex.

Get in Touch with GenScript CRISPR Experts

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