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Home Support FAQs CRISPR/CAS9 FAQs

CRISPR/CAS9 FAQs

1. Q: A 20bp target sequence is needed with a NGG PAM seq. Shall the NGG be exactly immediately following the 3' of this 20bp sequence?
A: Yes, the NGG is located immediately next to the 3' end of the 20bp sequence
2. Q: How to design the 20bp target-specific sequence?
A: The 20bp target-specific sequence should precede NGG (PAM). Please BLAST the seed region (12bp PAM-proximal) of the 20bp target sequence to make sure it’s unique along the genome to guarantee specificity.
3. Q: How many target RNA sequences should I use for a genome editing project?
A: Due to un-predicable nature of gRNA, we recommend 2 and more gRNA targeting sequences to be designed to make sure that at least one targeting sequence will work.
4. Q: Both of the guide and donor plasmids need to be transfected into cells; So transfection may be a limiting factor.
A: You can find a good transfection reagent for your cells. For hard to transfect cells, many researchers use electroporation. You can also try our Magnetofection transfection reagent that is good for hard to transfect cells: http://www.origene.com/cdna/transfection.mspx
5. Q: For knocking down a target gene, I guess we don't need a donor plasmid, correct?
A: Without donor template DNA, the double stranded break will be repaired by NHEJ; unpredicted indels will be introduced. You will screen the deletions/insertions that cause frame shift
6. Q: Do you have the cas9 antibody?
A: We are making the Cas9 antibody. In our CRISPR/Cas9 vectors, Cas9 has a C-terminal Myc-DDK tag. DDK is the same as Flag. You can try OriGene’s anti-DDK antibody (SKU TA50011-100)
7. Q: If I want to use CRISPR/Cas9 to knock down a certain gene, what kind of negative control should I include as negative control?
A: You can use a scramble control, pCas-Scramble, SKU GE100003
8. Q: Can CRISPR/Cas9 be used to produce knockout Rat?
A: Yes, CRISPR/Cas9 can be used to make knockout rat. You can inject mRNA (gRNA and Cas9 mRNA) or plasmid DNA (target sequence cloned in pCas-Guide) into the zygotes, then transfer the zygotes into pseudo pregnant rat
9. Q: Can it be used to produce a mutation in adult mouse tissue like brain?
A: If the gene is specifically expressed in brain, then you can make mutated mice, similarly to the knockout mice but with the mutation provided in the repair donor DNA when you do co-injection into the zygotes
10. Q: How to avoid off target issue using CRISPR/Cas?
A: You can blast your target sequences. If the off-target sequences don’t have the PAM (NGG), then they won’t be targeted by CRISPR/Cas9. You also want to choose target sequences with mismatches in the 8-14 bp at the 3’ end of the target sequences. This way, the off-target issue can be decreased dramatically. For therapeutic purpose, you can use Cas9 nickase which only cuts one strand
11. Q: What is the limit to multiple gene disruption?
A: You can do multiplexes using CRISPR/Cas9 system. You can do co-transfection or co-injection several guide RNAs into your cells; so you will achieve multiple gene disruption or genome editing. The limit could be transfection efficiency. Regarding how many genes you can do, it is not known yet
12. Q: How to screen the edited cells after transfecting the CRISPR/Cas9 vector?
A: If it is gene knockout, you can do WB to verify it. You can also do genomic PCR to detect the genomic integration. For mutations, you can amplify the genomic sequence, then clone the PCR fragment and sequence it. If you do gene knockout, the selection marker in the donor template DNA will help the selection. You will need to isolate individual cell colonies for introduction of specific mutations in the genome and other applications
13. Q: How do you make sure that Cas9 will not integrate in genome if you use lentivector?
A: For screening purpose, for short term, integration of Cas9 into the genome for 2 weeks seems to be ok. There is also a non-integration lenti packaging kit commercially available from Clontech
14. Q: Do you need to linearize a donor template before transfection for efficient repair?
A: The donor template DNA does not need to be linearized. For short insertion/deletion or mutations, you can use oligos as donor template DNA
15. Q: How to select for positive clones if using long oligos as a donor template?
A: Isolate single cell colonies, do WB (for gene knockout or tagging) or genomic PCR or sequencing (for mutations) to detect the genome editing depending on the nature of the editing
16. Q: Can we buy predesigned donor vectors?
A: Yes
17. Q: Does Crispr/Cas system work for non-dividing cells?
A: NHEJ repair works in non-dividing cells; HDR is not active in non-dividing cells. Therefore, you can do gene disruption using CRISPR/Cas9 system without donor template DNA
18. Q: For gene targeting in mice, do you recommend transfecting ES cells or pronuclei?
A: You can do both. You can inject mRNA (gRNA and Cas9 mRNA) or plasmid DNA (target sequence cloned pCas-Guide) into the zygotes or ES cells
19. Q: Can you introduce mutations anywhere in the genome, including in promoters or enhancers?
A: Yes. The 20 bp target sequences only need to precede NGG
20. Q: How long should LHA and RHA be?
A: 500-1000 bp should be enough for the left or right homologous arms for HDR mediated repair
21. Q: Do you know the specific cleavage site of the Cas:gRNA complex in terms of where in the targeting sequence the cleaving occurs?
A: Cas9 cleaves at 3 bp away from the 3’ end of the target sequence in the genome.
22. Q: Is there a method for cloning knockout cell lines from the engineered pool of cells?
A: Isolate individual cell colonies.
23. Q: Do you see variability in success with different cell lines?
A: Yes, depending on the cell line and target sequences.
24. Q: For fluorescently labeling a gene of interest, is it necessary to serial dilute transfected cells for clonal analysis?
A: If in the donor construct the fluorescent protein does not have a mammalian expression promoter, then you can sort the fluorescent cells out; you might not need to get individual cell colonies. If the fluorescent protein in the donor template DNA does contain a mammalian expression promoter, you will need to pass the transfected cells several generation to dilute the cells containing the donor construct expressing the fluorescent protein.
25. Q: What is the known efficiency relative to other genome editing approaches?
A: In general, the genome editing efficiency of CRISPR/Cas9 is similar or higher than TALEN. However, CRISPR/Cas9 is much more simple and easy to do. You will need to engineer the protein to recognize new DNA sequence in TALEN system, while CRISPR/Cas9 is RNA based.
26. Q: Why you need T7-drived vector to express gRNA and cas-9?
A: For making gene knockout mice and genome editing in other organisms, such as Drosophila, some researchers do microinjection of gRNA and Cas9 mRNA into cells

 

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