RiboBio offers a convenient and efficient gene editing package: efficiency Guarantee set and standard set. The Efficiency Guarantee Set is tailored to human-derived genes, designed to select 5 crRNAs with high scores, and is configured with essential reagents based on Cas9 mRNA systems, including tracrRNA, Cas9 mRNA, mRNA transfection reagents, positive control crRNA validation set, T7E1 enzyme and supporting buffers, etc., providing a ready-to-use gene editing system. The efficiency guarantee set can realize the T7E1 digestion efficiency of MHCC97H cells to at least 1 crRNA reaching 30% or more and provide corresponding after-sales service. The standard set does not provide any editing efficiency guarantee, while with the same reagents as the guarantee set.

riboEDIT™ CRISPR-Cas9 Set

*Efficiency Guarantee Set: Based on the instructions, the T7E1 digestion efficiency of MHCC97H cells can realize at least 1 crRNA reaching 30% or more. If 5 crRNAs are all less than 30%, the customer must provide MHCC97H transfection efficiency and detection method steps. After technical analysis, it is free to re-optimize the design, select, synthesize 5 crRNAs and provide experimental guidance. See the manual for more information.

**Positive Control crRNA Validation Set for Human contains human positive control crRNA (5nmol), forward primer (5nmol) and reverse primer (5nmol).

riboEDIT™ Designed crRNA adopts RiboBio’s optimized bioinformatics design, which requires no crRNA/sgRNA design experience for clients. crRNA contains 20 complementary pairing nucleotides (original spacer) for the target DNA fragment and complementary paring repeated sequence for tracrRNA, which covers human and mouse gene. Each gene will be 3-6 individual sgRNAs through strict PAGE purification. If you need design crRNA for other species, please evaluate accordingly.

The reference price for human and mouse gene: 5nmol, 1250rmb for one crRNA.

Positive Control crRNA Validation Set is used to detect the correctness of the gene cleavage system for Human, including human positive control crRNA (5nmol), forward primer (5nmol) and reverse primer (5nmol).

riboEDIT tracrRNA is chemically synthesized tracrRNA, which is purified by PAGE and is resistant to nucleases. It can bind to crRNA to form a crRNA-tracrRNA complex, which together guides Cas9 protein to cleave double-stranded DNA.

riboEDITTM Cas9 mRNA is the in vitro transcribed Cas9 mRNA with cap and poly A tail. Coding vector is human condon optimized S. pyogenes Cas9, which includes NLS, 1XFLAG tag at C terminal, 5’UTR and 3’UTR to improve mRNA translation and increase mRNA stability. After strict QC, the quality and purification are guaranteed, which is applicable to mammalian cells gene editing.

riboEDIT™ Cas9 Expression mRNA with High-efficient Genome Cleavage Efficiency

Figure 1 Transfect MHCC97H cells with 10pmol riboEDIT™ crRNA, 10pmol riboEDIT™ tracrRNA and 1ug riboEDIT™ Cas9 mRNA. After 48 hours transfection, there will be riboEDIT™ T7EI Enzyme cleavage efficiency for target site, and cleaved bands and editing efficiency for 7 candidate target genes. As shown above, #1#2 represents same gene with 2 crRNAs for different sites.

riboEDIT Cas9 RNP with High-efficient Genome Cleavage Efficiency

Figure 2 Transfect MHCC97H cells with 6pmol riboEDIT™ crRNA, 6pmol riboEDIT™ tracrRNA and 6pmol riboEDIT™ Cas9 Protein. After 48 hours transfection, there will be riboEDIT™ T7EI Enzyme cleavage efficiency for target site, and cleaved bands and editing efficiency for 7 candidate target genes. As shown above, #1#2 represents same gene with 2 crRNAs for different sites.

Figure 3 riboEDIT™ CRISPR-Cas9 mRNA gene editing system and riboEDIT Cas9 RNP complex have equivalent cleavage activity for target site.

1. What advantages does the riboEDIT CRIPSR-Cas9 system have over the Cas9 stable expressing cell lines or lentiviral systems, or Cas9 plasmid vectors?

A: The main advantages are as follows:

(1) Total RNA system of riboEDIT™ CRIPSR-Cas9 or Cas9 protein system, comparing with plasmid expression vector or Cas9 stable expressing cell lines, Cas9 protein system is instant expression with low off-target effect, no DNA integration risk and promoter compatibility, which hugely improves the application safety.

(2) Ready-to-Use. One step transfection and complete the editing efficiency detection within 5-6 days with easy operation and shorter experimental period.

(3) The RNP system rapidly undergoes gene editing post 12-24 hours transfection, and the plasmid vector generally takes 72 hours.

(4) No need to self-construct vector or pack lentivirus as well as virus level laboratory.

(5) It has higher gene-editing efficiency comparing with plasmid expression vector in most of cells.

(6) For most cells, multiple targets in a cell can be edited by transfecting multiple crRNAs without constructing a multi-target CRISPR expression vector, introducing no extra expression elements or resulting cytotoxicity.

2. What system should we choose from riboEDIT™ CRIPSR-Cas9 for stem cells, primary cells or suspension cells?

A: Stem cells, primary cells or suspension cells are difficult to transfect with cells and transfection reagent usually cannot reach the ideal transfection result. Electroporation is more suitable for these kinds of cells. Based on current references, Cas9 RNP system is used for electroporation which will have higher gene editing efficiency. For the above cells with difficult transfection, riboEDIT™ CRISPR/Cas9 RNP system is recommended. Specific experimental conditions can be adjusted accordingly.

3. How to quickly confirm the crRNA effiency??

A: crRNA/tracrRNA cleavage efficiency is related to target sequence identified by crRNA. Different crRNA will have various cleavage activities. In vitro enzyme cleavage experiment is recommended. In vitro, cleave target DNA fragment by enzyme, rapidly screen the effective crRNA, obtain crRNA with high editing efficiency and perform the downstream experiment.

4. What is the PAM sequence identified by riboEDIT^TM CRISPR-Cas9 gene-editing system?

 A: riboEDIT^TM Cas9 mRNA and riboEDIT^TM Cas9 Protein are S. pyogenes Cas9, and the identified PAM sequence NGG. N represents A, T, C, G.

5. How to guarantee the low off-target effect for riboEDIT^TM Pre-designed crRNA?

A: From the design perspective of crRNA, using more stringent sequence alignment analysis, Ribobio provides professional leading crRNA design synthesis custom service!

6. What are the reasons if there are no cleaved bands when using T7E1 enzyme cleavage to detect editing efficiency?

A: The possible reasons are as follows:

(1) Transfection efficiency for cells is low. It is recommended to optimize transfection steps or change to cell types that are easily transfected.

(2) For Cas9 mRNA or Cas 9 protein degradation, positive control can be set to eliminate the Cas9 mRNA or Cas9 protein degradation issues.

(3) Cas9 nuclease cannot reach or cleave target site in cells, it is recommend to redesign crRNA.

(4) There are no denaturation and annealing for DNA fragment. Set positive control to ensure if the experimental operation is correct.

7. What are the solutions if there are many non-specific bands when using agarose gel electrophoresis to analyze cleavage efficiency?

A: Set negative control to differentiate target shear bands and non-target bands. If necessary, redesign primer or optimize PCR conditions to lower non-specific amplification. It is recommended to use nested PCR to improve the specificity of amplified fragment.

8. What are the solutions if there are band dispersion when perform T7E1 mutation detection?

A: T7E1 has weak ability of non-specific cleaving DNA strand, therefore, you can increase DNA dosage, lower enzyme dosage and reduce enzyme cleavage time to decrease non-specific cleavage situation.

9. What does the riboEDIT CRISPR-Cas9 Gene Editing Efficiency Guarantee set include? Where can I order?

A: The Efficiency Guarantee Set is based on the whole RNA system providing crRNA, tracrRNA, Cas9 mRNA, mRNA transfection reagent, positive control crRNA validation set, T7E1 enzyme and supporting buffer. The set of 5 crRNAs in the set are optimized. The Efficiency Guarantee Set provides more after-sales service. You can order all products on www.ribobio.com.

10. Is it possible to transfect multiple crRNAs to achieve editing of multiple targets or genes?

A: Yes. Mixing multiple crRNAs, co-transfection with tracrRNA and Cas9 mRNA (or Cas9 protein) can be used to edit multiple targets or genes. The best co-transfection system needs to be optimized by itself. Please ensure add crRNA and tracrRNA in ratio of 1:1.

11. If you want to use the crRNA library for screening, do you have to match the Cas9 mRNA or protein provided by RiboBio?

A: The PAM sequence recognized by synthetic crRNA is NGG (N stands for A, T, C, G), which can be used in combination with S. pyogenes Cas9 plasmid or S. pyogenes Cas9 stable cell line. Due to editing efficiency, it will be affected by the expression of Cas9 protein. Therefore, the crRNA/tracrRNA designed by Ribobio does not have to be used with Cas9 mRNA or protein provided RiboBio, but in the case of other non-RiboBio Cas9 mRNA or protein, high editing efficiency conditions need to be explored or tested by itself.