Re: knock down large fragments using CRISPR/Cas9

[Faheem Kanwal]

Hi Lina, 

1. Knock-down?   For knock-down we usually use the dCas9, which specifically bind to active site of enhancer where transcription factors  binds. if you have Chip-seq or ATAC - data..otherwise .. you  can knock-down through 12kb  deletion then Cas9 is ok, 

2. For dCas9, design gRNA at or near transcription binding site, for deletion design at least  4 to 5 gRNA at flanking sites of 12 kb ( we did one time, it worked well)  , then test the gRNA efficiency by T7 endo nuclease essay, like first inject gRNA with Cas9,( RNP)  then amplify it with PCR and do T7 endonuclease ... if gRNA working.. you will get TWO bands  on gel after T7 endonuclease ... 

3. you can use IDT(https://www.idtdna.com/site/order/designtool/index/CRISPR_SEQUENCE) so for best for my all gRNAs.

4. you can directly use the RNP(gRNA and Cas9/dCas9), we usually gets  best results with RNP ( i only use vector for Knock-in) 

Best, 

Kanwal 

On Tuesday, April 29, 2025 at 2:44:02 PM UTC-4 lina shi wrote:

Dear Professor

I want to knock down a 12kb super enhancer from zebrafish genome using CRISPR/Cas9, could you please give me some advice about how to do it? Like which enzyme do I need to use? How many gRNA do I need to design? Which tool can I use to design gRNA? Which location do I need to target to delete the super enhancer? Which expression vector can I use? I am looking forward to your reply! Thanks so much in advance!

Best

Lina

[haruna daniel]

Dear prof kanwal can you share your protocol on RNP thanks

--
You received this message because you are subscribed to the Google Groups "Genome Engineering using CRISPR/Cas Systems" group.
To unsubscribe from this group and stop receiving emails from it, send an email to crispr+un...@googlegroups.com.
To view this discussion visit https://groups.google.com/d/msgid/crispr/cedbee6a-0cb0-498b-8892-c6dcb7610aebn%40googlegroups.com.

[Faheem Kanwal]

Hi Lina, 

1. Design two gRNA pairs and test each one at the flanking sites. Make sure to try deleting the entire region. Partial deletions can sometimes trigger RNA compensation mechanisms, as reported by Didier Lab (Nature, 2019). To avoid this, aim to delete the maximum possible sequence. After designing the gRNAs, validate them using the T7 endonuclease assay. Proceed with the two gRNAs that show the highest editing efficiency. With just two gRNAs, we successfully deleted a full 12 kb region.

2. Use RNP complexes for deletion or knock-out experiments. Based on  literature review and many KO studies, expression vectors are not effective for this purpose specifically in zebrafish . Instead, use two highly efficient gRNAs targeting the flanking regions along with Cas9 protein. This RNP approach consistently yields the best results in zebrafish models.

3. Avoid using expression vectors—use RNPs instead. Many published studies have successfully used RNPs for injection. In our lab, we typically inject 1 µL of gRNA and 1 µL of Cas9 protein (optimized protocol). For your experiment, inject 2 µL of each flanking-site gRNA and 4 µL of Cas9 protein at the one-cell stage.

4. Our lab does not recommend vectors for knock-out studies. Stick to RNPs for better efficiency and reliability.

Good luck 

Best, 

Kanwal 

On Wed, Apr 30, 2025 at 3:14 PM lina shi <shili...@gmail.com> wrote:

  Dear Prof. Kanwal,  

Thank you very much for your response.

I plan to delete a 12 kb region using CRISPR/Cas9. From your suggestion, I understand that I should design 4–5 gRNAs targeting each flanking region of the 12 kb segment—meaning a total of 8 to 10 gRNAs. Is that correct?

If so, I have a few follow-up questions:

       1. At each flanking site, what range should I consider for designing the gRNAs? Should they be designed within a 1 kb region, or is there an optimal window you would recommend to ensure efficient deletion? 

       2. Is it feasible to express that many gRNAs along with Cas9 from a single expression vector?

       3. If not, is it standard practice to mix multiple gRNA-expressing vectors together for injection?

       4. Since we are doing this in zebrafish, do you have any recommended vectors or systems that we could order for this purpose?

Thank you for your guidance.

Best regards,

Lina

--

You received this message because you are subscribed to the Google Groups "Genome Engineering using CRISPR/Cas Systems" group.
To unsubscribe from this group and stop receiving emails from it, send an email to crispr+un...@googlegroups.com.

To view this discussion visit https://groups.google.com/d/msgid/crispr/882bab16-7d0f-4601-bcea-81891ff2dd80n%40googlegroups.com.

[lina shi]

Thanks so much,  Prof. Kanwal!!!

[Faheem Kanwal]

Hi Lina, 

Welcome, I hope it will work best for you. 

Good Luck 

To view this discussion visit https://groups.google.com/d/msgid/crispr/79eac6c0-89c1-4eb5-909b-4848668b8517n%40googlegroups.com.

[Faheem Kanwal]

HI Haruna, 

To prepare a 10 µL injection mix, use the following components based on a 1:2 ratio of gRNA to Cas9 protein:

• 2 µL of guide RNA (gRNA)

• 4 µL of Cas9 protein (maintaining the 1:2 gRNA:Cas9 ratio)

• 4 µL of phenol red or an appropriate buffer for injection tracking

This mix ensures optimal RNP complex formation and visibility during microinjection.

Best, 

Kanwal