The riboEDIT CRISPR-Cas9 system developed by RiboBio is using the natural complex system (crRNA and tracrRNA). RiboBio provides a Cas9 mRNA-based total RNA system and a Cas9-based RNP system. The tracrRNA can be synthesized on a large scale. The crRNA targeting the DNA sequence can be synthesized by high-throughput, and can be simply transfected or electrotransformed into mammalian cells. It is a ready-to-use system, which does not require self-construction of gRNA expression vector, no requiring of manipulation in a virus-scale laboratory, without any DNA components, and can achieve multi-target editing by the guidance of multiple crRNAs, thus greatly simplifying the preparation of preliminary experiments. Steps. This system is more suitable for gene knockout screening at high throughput levels. RiboBio also offers industry-leading CRISPR-Cas9 editing efficiency gurantee set to realize a convenient, time-saving, reliable and efficient solution for genetic editing experiments.
RiboBio Patent: A system for DNA editing and its application, Chinese patent application number: 108977442A, PCT application number: CN2018/088105
1. RiboBio patented technology: optimized crRNA, tracrRNA and Cas9 mRNA
2. Transient expression, effectively reducing the off-target rate
3. High editing efficiency and can target multiple DNA sequence once a time
4. Less toxic, less innate immune response
5. Ready-to-use system, easier to use, more suitable for small-scale experiments to large-scale high-throughput screening
6. No need for cumbersome cloning process, saving more manpower and time
7. No need for virus-level laboratories, greatly reducing the requirements of genetic editing for the experimental environment
riboEDIT™ Editing Efficiency Guarantee Set & Standard Set
RiboBio offers a convenient and efficient gene editing package: efficiency assurance set and standard set. The Efficiency Assurance 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
|Name||riboEDIT CRISPR-Cas9 mRNA Set, 20T|
|The efficiency guarantee set *||No.：crG00001|
|The Standdard Set||No.：crS00001|
*Efficacy Guarantee: Under the operating conditions of the instructions, the T7E1 digestion efficiency of MHCC97H cells can reach 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 and synthesize 5 crRNAs and provide experimental guidance. See the manual for more information.。
|Name||Components & Reagents|
|riboEDIT CRISPR-Cas9 mRNA Set||riboEDIT™ Designed crRNA（设计合成5条crRNA，每条5nmol）
riboEDIT™ tracrRNA， 5nmol
riboEDIT™ Positive Control crRNA Validation Set for Human *, 3*5nmol
riboFECT™ mRNA Transfection Reagent, 75ul
riboEDIT™ Cas9 mRNA (0.5ug/ul), 20ug
riboEDIT™ T7EI Enzyme (10U/ul), 100U
**Positive Control crRNA Validation Set for Human including positive control crRNA 5nmol for human, forward primer 5nmol and reverse primer 5nmol.
Designed and Synthesized crRNA
riboEDIT™ Designed crRNA采用锐博生物优化的生物信息学设计，客户无需具备crRNA/sgRNA设计经验，crRNA包含20个与目标DNA序列互补配对的核苷酸（原间隔序列）和与tracrRNA互补配对的重复序列，涵盖人类和小鼠基因，每个基因设计合成3 – 6段单独的sgRNA，经过严格的PAGE纯化，其他物种crRNA设计需另行评估。
Positive Control crRNA Validation Set
Positive Control crRNA Validation Set is used to detect the correctness of the gene shear system for Human, including positive control crRNA 5nmol for human, 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.
riboEDIT Cas9 mRNA is generated in vitro and has a cap and polyA tail structure. The coding sequence is a human codon-optimized S. pyogenes Cas9 with a nuclear localization signal (NLS), a 1XFLAG tag at the C-terminus, and a 5′ untranslated region ( 5’UTR) and 3′ untranslated region (3’UTR) to promote mRNA translation and improve mRNA stability, suitable for mammalian cell gene editing.
Examples: riboEDIT™ CRISPR-Cas9 mRNA与RNP Systems
riboEDIT™ Cas9 Expression mRNA具有高效的基因组剪切效率
图1. MHCC97H细胞共转染10pmol riboEDIT™ crRNA、10pmol riboEDIT™ tracrRNA和1ug riboEDIT™ Cas9 mRNA，48小时后 riboEDIT™ T7EI Enzyme酶切检测靶位点的剪切效率，候选7个靶基因的剪切条带及编辑效率。如图所示，#1#2表示同一基因不同位点设计的2条crRNA。
riboEDIT Cas9 RNP具有高效的基因组剪切效率
图2. MHCC97H细胞共转染6pmol riboEDIT™ crRNA、6pmol riboEDIT™ tracrRNA和6pmol riboEDIT™ Cas9 Protein，48小时后 riboEDIT™ T7EI Enzyme酶切检测靶位点的剪切效率，候选7个靶基因的剪切条带及编辑效率。如图所示，#1#2表示同一基因不同位点设计的2条crRNA。
图3. riboEDIT™ CRISPR-Cas9 mRNA基因编辑体系与riboEDIT Cas9 RNP复合物具有等同的靶位点剪切活性。
1. What advantages does the riboEDIT CRIPSR-Cas9 system have over the Cas9 stably expressing cell lines or lentiviral systems, or Cas9 plasmid vectors?
A: The main advantages are as follows:
(1) riboEDIT CRIPSR-Cas9 total RNA system or Cas9 RNP system Compared to plasmid vector or Cas9 stably expressing cell line, Cas9 protein is transient expression, low off-target efficiency, no DNA integration risk and promoter compatibility issues. Improve application security.
(2) Ready-to-use system, through one-step transfection, 5-6 days can complete the editing efficiency detection, the operation is simple, and the experimental period is significantly shortened.
(3) The RNP system rapidly undergoes gene editing after 12-24 hours of transfection, and the plasmid vector generally takes 72 hours.
(4) There is no need to construct a vector or package a lentivirus, and no virus-level laboratory environment is required.
(5) In most cells, it has higher gene editing efficiency than the plasmid expression vector.
(6) In 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. Which type of riboEDIT CRIPSR-Cas9 system is selected for performing such as stem cells, primary cells or suspension cells?
A: Stem cells, primary cells or suspension cells are difficult to transfect cells. Transfection reagents are generally difficult to achieve the desired transfection effect. The electroporation method is more suitable for transfection of such cells. According to the current literature, Cas9 RNP system is used. Electrotransfer has higher gene editing efficiency. For the above-mentioned difficult-to-transfect cells, riboEDITTM CRISPR-Cas9 RNP system is recommended, and the specific experimental conditions need to be optimized.
3. How to quickly determine the validity of crRNA?
A: The efficiency of crRNA/tracrRNA cleavage is related to the target sequence recognized by crRNA. Different crRNA cleavage activities are different. It is recommended that the target DNA fragment can be digested in vitro by CRIPSR-Cas9 system in vitro, and the effective crRNA can be rapidly screened. Highly active crRNA is then subjected to cell level experiments.
4. What is the PAM sequence identified by the riboEDIT CRISPR-Cas9 gene editing system?
A: riboEDIT Cas9 mRNA and riboEDIT Cas9 Protein are S. pyogenes Cas9, which recognizes the PAM sequence NGG, and N stands for A, T, C, G.
5. How does riboEDIT Pre-designed crRNA ensure low off-target efficiency?
A: From the design perspective of crRNA, using more stringent sequence alignment analysis, Ruibo Bio provides professional leading crRNA design synthesis custom service!
6. T7EI enzyme digestion detection editing efficiency found out what is the cause of no shear band?
A: The possible reasons are as follows:
(1) Cell transfection efficiency is low, it is recommended to optimize the transfection step or switch to a cell type that is easily transfected.
(2) Cas9 mRNA or Cas9 protein degradation can be ruled out by setting a positive control group to exclude Cas9 mRNA or Cas9 protein degradation.
(3) In the cell, the Cas9 protein cannot access the target site or the target site cannot be sheared. It is recommended to redesign the crRNA.
(4) There is no denaturation or annealing step of the DNA fragment. The experimental operation can be confirmed by setting a positive control group.
7. Agarose gel electrophoresis analysis of shear efficiency when there are many non-specific bands, can not analyze the shear efficiency?
A: Target cut bands and non-target bands can be distinguished by setting a negative control group. Primers need to be redesigned if necessary, or by optimizing PCR conditions to reduce non-specific amplification. Nested PCR is recommended to increase the specificity of amplified fragments.
8. When the T7E1 mutation is detected, the band dispersion phenomenon occurs. How should it be solved?
A: Because T7E1 itself has a weak ability to non-specifically cleave DNA strands, this can increase the amount of DNA, reduce the amount of enzyme, and reduce the time of digestion to reduce non-specific cleavage.
9. What does the riboEDIT CRISPR-Cas9 Gene Editing Efficiency Assurance Package include? Where can I order?
A: The efficiency assurance set is based on the whole RNA system and provides 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. Edit efficiency guarantee set provide more after-sales service. You can order all products on the homepage of www.ribobio.com.
10. Is it possible to transfect multiple crRNAs? To achieve editing of multiple targets or genes?
A: Yes, by 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 crRNA and tracrRNA. added in 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, but due to editing efficiency Will be affected by the expression of Cas9 protein. Therefore, the CRRNA/tracrRNA designed by Ruibo 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.
|C11062-1||riboFECT mRNA Transfection Control(EGFP),500ng/μL, 10ug||$199.92|
|C11057-2||riboEDIT Cas9 mRNA (0.5ug/ul), 40ug||$902.97|
|C11057-3||riboEDIT Cas9 mRNA (0.5ug/ul), 100ug||$1,832.60|
|crG00001||riboEDIT CRISPR-Cas9 mRNA Guarantee Set, 20T||$2,663.93||-||Start Customization|
|crS00001||riboEDIT CRISPR-Cas9 mRNA Standard Set, 20T||$1,997.53||-||Start Customization|
|CR-NRA-1||riboEDIT Designed crRNA||Inquiry||-||Start Customization|
|crRP0001VS||riboEDIT Positive Control crRNA Validation Set for Human||$166.60|
|crT00001-5||riboEDIT tracrRNA, 5nmol||$104.62|
|crT00001-20||riboEDIT tracrRNA, 20nmol||$229.90|
|C11055-1||riboFECT mRNA Transfection Reagent, 75ul||$133.28|
|C11057-1||riboEDIT Cas9 mRNA (0.5ug/ul), 20ug||$491.47|
|C11053-1||riboEDIT Cas9 Protein, S.pyogenes(20uM), 500pmol||$209.91|
|C11054-1||riboEDIT T7EI Enzyme (10U/ul)，100U||$146.60|
Custom Oligo Synthesis Click for custom Oligos