CRISPR/Cas9 sgRNA Synthesis

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 crispr@genscript.com.

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 crispr@genscript.com.

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 crispr@genscript.com.

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 crispr@genscript.com.

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.

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.