SygRNA® Synthetic gRNA and sgRNA Design

Frequently Asked Questions

What is the difference between modified and unmodified guide RNAs?

Modified guide RNAs have a chemically modified backbone by adding phsphorothioate linkages to the 3' and 5' ends of the guide RNA as well as stabilizing 2'O-methyl bases whereas unmodified gRNAs do not.

How do I choose between modified and unmodified gRNAs?

For easy-to-transfect cell lines, we suggest unmodified/desalted. For difficult-to-transfect cell lines such as primary cells, we recommend modified/HPLC-purified. This is because modifications have been shown to improve stability and protection from exonucleases and can improve editing efficiency. This is particularly beneficial in difficult to transfect cell lines but can be a benefit in all experiments.

Can I use your synthetic crRNA on its own?

No, a two-part guide RNA requires both crRNA and tracrRNA. The tracrRNA recruits the Cas9 protein and is an essential part of a ribonucleoprotein (RNP) complex.  An alternative option is to use our single guide RNA products.
Please note that the unmodified crRNA requires the unmodified tracrRNA and the modified crRNA requires the modified tracrRNA..

Why do I need the tracrRNA?

The standard CRISPR system comprises three components: crRNA (targeting RNA), tracrRNA (trans-acting crRNA) and SpCas9. For the system to function, all three components must be present. SygRNA® tracrRNA is optimized for use with SygRNA® crRNA.

Should I anneal my crRNA and tracrRNA?

With crRNA, tracrRNA and Cas9 protein added all at once, no prior annealing step is necessary. See our detailed protocols at SigmaAldrich.com/SygRNAprotocols

Will your crRNA function correctly with a tracrRNA from another source?  

tracrRNA from various sources can have differences in length or sequence; our tracrRNA has been optimized for use with our crRNA. Therefore, we recommend using our crRNA with our tracrRNA together for best results.

What controls do you offer, and which should I use?

We offer universal negative controls for human, mouse and rat species as well as positive controls for human and mouse which can be found here. For custom control please contact us for a custom quotation. The sequences for our controls are confidential.

When should I use desalted or HPLC-purified gRNAs? 

For cell lines, we recommend desalt. If working with animal models, we recommend HPLC as this is a purer product to microinject/electroporate.

Do you offer single guide RNAs (sgRNA) instead of a two-part system?

Yes, we offer both modified and unmodified single guide RNAs (sgRNAs).

How do I determine if I should use the one-part sgRNA or the two-part crRNA/tracrRNA system?

Both provide efficient cutting at your desired target site for standard applications. However, in many experiments the one-part sgRNAs have been shown to have higher activity.

Do you sell Cas9 proteins?

Yes, we offer a wide variety of Cas9 proteins for any gene editing application. Our wild-type SpCas9, high fidelity eSpCas9 and additional Cas9 proteins can be found here.

Do you have any protocols for complexing the synthetic guide RNAs?

Yes, visit SigmaAldrich.com/SygRNA for recommended transfection and nucleofection protocols in cell lines, T cells, iPSCs, animal embryos, and more.

Do you offer a guide RNA design service?

Yes, there is a free online gRNA design tool here. If you would like design assistance, we offer a free design service for guideRNAs. Please contact CRISPR@sial.com.

Do you manufacture donor constructs?

Donor oligos up to 120 bases may be ordered here. For donor oligos between 121 – 180 bases, please contact CRISPR@sial.com.

How long should my gRNA target sequence be?

To optimize efficiency and specificity, our gRNA target sequences are 19 bases, excluding the PAM sequence. We routinely accommodate requests to manufacture guides with targets from 18-20bp.

Why should the PAM site not be included in my target sequence when ordering?

The PAM is a sequence adjacent to the genomic target and is not a part of the gRNA. For ordering we request you enter only the 18-22 nucleotides of gRNA sequence to be synthesized.

What is your turnaround time?

You should expect to receive sgRNA within 7-10 business days and crRNA/tracrRNA in 3-5 days from the time of order.

Do you offer custom quantities of crRNA and tracrRNA?

Yes, please contact us at CRISPR@sial.com to submit your request.

Do you offer labelled gRNA?

Yes, we offer a variety of crRNA modifications including fluorescent dyes.  Please send a request to CRISPR@sial.com.

Do you offer labelled tracrRNA?

Yes, we offer select fluorescent dyes including Cyanine3, Cyanine5, ATTO488, ATTO590, which are conjugated to the 5’ end of the tracrRNA. Please send a request to CRISPR@sial.com.

Is there an enrichment method with synthetic CRISPR?

The fluorescently labelled tracrRNAs above are suitable for use in FACS-based enrichment of CRISPR-containing cell populations.  To order, please click here

How many guides should I test per target/gene to ensure optimal efficiency?

We recommend testing at least three gRNAs per target/gene. Please note, if using nickase protein, pairs of guide RNAs will need to be tested.

Should I use multiple gRNAs simultaneously to knockout a single gene?

It is possible that using multiple guides could increase the cutting efficiency at the target site, but it also increases the risk of unexpected or unwanted deleterious effects. We do not currently recommend using more than one crRNA or sgRNA per target.

How do I reduce off-target effects?

SpCas9D10A and eSpCas9 protein have been demonstrated to reduce off-target effects compared to WT SpCas9. Additionally, RNP is thought to reduce the chance of off-target effects as compared to plasmid-based CRISPRs by shortening the time that CRISPR is present in the cell.

What is the difference between WT SpCas9 protein, eSpCas9, and SpCas9 nickases?

WT SpCas9 functions with one guide RNA to introduce a double strand break, but this first-generation protein carries some risk for off-target effects. To achieve higher-fidelity genome editing, the simplest approach is to use eSpCas9, coupled with one guide RNA. An alternative approach to achieving higher specificity is the SpCas9D10A nickase. Nickase requires two individual gRNAs for cleavage and and has the additional feature of creating “sticky-end” overhangs at the site of the double strand break.  These sticky ends may have additional utility in experiments where overhangs promote donor construct integration. Learn more about T cell editing with nickase RNPs here

How does dead SpCas9 differ from other Cas9 proteins?

Our dCas9-3XFLAG™-Biotin protein is mutated to be nuclease-inactive. It is essentially a tether for other functional groups in applications including CRISPR activation and inhibition, chromosome isolation via ChIP or chromosome detection methods such as FISH.  It is also an essential component of the proxy-CRISPR system. Read more here.

Can I use FnCas9 for genome editing?

There are some useful applications for FnCas9 in genome editing, such as the dual Cas9 proxy-CRISPR system.  However, due to the inherent low activity this protein is useful primarily in “cell-free” applications in synthetic biology.

Can I use SpCas9 plasmid with synthetic gRNA instead of Cas9 mRNA or protein?

Yes. However, when using a plasmid, there is a lag in activity while transcription and translation occur, and some of the synthetic gRNA may be susceptible to degradation. Therefore, we recommended combining synthetic gRNA with Cas9 mRNA or protein.

What quantity of sgRNA/crRNA/tracrRNA/SpCas9 protein am I meant to use?

Visit SigmaAldrich.com/SygRNA for protocols. Please note that these protocols are meant as a guide, and individual experiments may need to be optimized accordingly.

Why is my CRISPR genome editing efficiency low and how can I improve it?

There are multiple factors to consider when optimizing efficiency of any genome editing experiment.

Assay - Genome editing efficiency may be assessed by a variety of methods, each varying in sensitivity from the next.  In general, mismatch assays such as CEL1 or T7E1 may underrepresent cleavage efficiency, because they are not as sensitive as sequencing-based methods such as TIDE, NGS and other methods.

Delivery – Optimization of delivery method is also critical and should be evaluated for each cell line prior to initiation of genome editing experiments. Liposomal transfection, electroporation and lentiviral transduction are all efficient modes of delivery when optimized.

DNA structure - While SpCas9 accesses most chromosomal targets efficiently, chromatin structure may influence cleavage efficiency from one genomic site to another, or one cell line to another.  Testing more than one gRNA optimizes for the highest efficiency.

What controls do you offer, and which should I use?

We offer universal negative controls for human, mouse and rat species as well as positive controls for human and mouse which can be found here. For custom control please contact us at CRISPR@sial.com for a custom quotation. The sequences for our controls are confidential.

How can I verify that my gene has been knocked out?

At the molecular level, DNA, RNA, and protein assays may all reveal gene disruption.  We strongly suggest, however, that a DNA-based assay be performed first to avoid confusing results from downstream variables, both within the cell and as part of the experimental assay. Suitable assays include CEL1/T7E1, RFLP, direct Sanger sequencing, NGS, DNA fragment analysis and TIDE.

I can still detect a band in my western blot.  Does this mean my gene is not knocked out? 

Not necessarily.  False positives are common due to non-specific binding of antibodies.  For the most reliable detection of gene knockout perform one of the DNA-based assays listed above.

What is the stability of your Cas9 proteins?

As long as the proteins remain lyophilized we guarantee activity for 2 years from the date listed on the CoA.