Frequently Asked Questions: SygRNA® Synthetic CRISPR

How do I choose between modified and unmodified crRNAs? 

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. Most animal model researchers prefer unmodified/HPLC-purified gRNAs. 

Can I use your synthetic crRNA on its own? 

No, a two-part guide RNA requires both crRNA and tracrRNA. The tracrRNA recruits 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 to function properly. 

Why do I need the tracrRNA? 

The wild-type (WT) CRISPR system comprises three components: crRNA (targeting RNA), tracrRNA (trans-acting crRNA) and SpCas9 protein. 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? 

Our protocols at SigmaAldrich.com/SygRNA describe one step complexing; crRNA, tracrRNA and Cas9 protein added all at once, no prior annealing step necessary. 

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 for best results.

When should I use desalted or HPLC crRNA?  

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 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 variety of Cas9 proteins. 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. For microinjection protocols for mice and zebrafish, please contact us at crRNA@sial.com for protocols shared by customers.

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 guide RNAs. Please contact crRNA@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 frequest@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 18-22bp.

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? 

One-part sgRNAs ship in 7-10 business days and two-part crRNA/tracrRNA ships in 3-5 business days.

Do you offer custom quantities of crRNA and tracrRNA?

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

Do you offer labelled crRNA? 

Yes, we offer a variety of crRNA modifications including fluorescent dyes.  Please send a request to crRNA@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 crRNA@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 crRNA or sgRNAs 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 guideRNA.  An alternative approach to achieving higher specificity is the SpCas9D10A nickase.  Nickase requires two individual gRNAs for cleavage 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. 

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 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 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 CELI 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. Please contact us crRNA@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 CELI/T7E1 (mismatch detection assay), restriction fragment length polymorphism (RFLP), direct Sanger sequencing, DNA fragment analysis, and TIDE: tracking of indels by decomposition.

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.