I have not done this kind of experiments, but these are my thinking/opinion:

1. You said " I am wondering if the manual is suitable for crRNA + tracrRNA, since normally for microinjections these are incubated with Cas9 at 37C for at least 15 mins before usage." But the instruction also said that after added to cells, you incubate it at 37C for 2 days. (see attachment, page 2, yellow highlight)

2. The website states that "Demonstrated cleavage efficiency—tested in over 20 cell types including iPSC, mESC, N2A, CHO, A549, HCT116, HeLa, HEK293, and several others". Do you know: has it been done on your Neuro2A cells? If you have other cell types they mention, maybe you can try one on them as a control?!

3. Do you think that the 'amount' ratio of (74ng tracrRNA + 54ng crRNA) can be a problem? Although the total amount is around 125 ng, as suggested by them.

By the way, I don't know whether you have seen this paper below (see attachment):

Title: "Improved delivery of Cas9 protein/gRNA complexes using lipofectamine CRISPRMAX"

Take a look at its Fig. 1b. Using lipofectamine CRISPRMAX, some cell types have good efficiency, but not for other cell types.

Thanks for responding Yuan-Yeu and Mark, 

Yuan-Yeu, after transfection I did wait 2 days before harvesting the cells to isolate genomic material. It has been tested in Neuro2A (N2A) cells as you wrote, and in any case I think transfection works (I added a GFP plasmid in the control and there was abundant GFP expression). I'm not sure if the amount is right, I will optimise that next. 

Mark, my aim is to produce a transgenic mouse with a tag inserted into the gen of interest. I've read several papers and it seems that insertion efficiency is improved when using crRNA + tracrRNA vs sgRNA, see the following paper by Aida et al., 2015, DOI 10.1186/s13059-015-0653-x. Thanks for letting me know about Mirus RNA transfection products. 

At the moment I'm testing whether pre-incubation of Cas9 with the RNAs @ 37C prior to transfection improves the experiment. 

Hi Amila,

I appreciate your response, and please do update us after you get new results. Thanks.

By the way, what is the reason make you think that "pre-incubation of Cas9 with the RNAs @ 37C" is better than "after you add them to the cells then incubate at 37C"? So, let them form a complex first?

Hi Yuan-Yeu, yes exactly. Pre-incubation is usually performed before microinjections so I reasoned that it may be necessary. I'll keep you posted!

Hi Mark,

1. It also mentions in the attached paper that 'the shortened sgRNA isn't as efficient since it's truncated, and several other papers show that extending the duplex by ~5 bp can increase efficiency...' (Page 2, yellow highlight).

2. So, in TransIT-X2®protocol you provided above,it also suggests to pre-incubate the components together at room temperature for 15-30 min to allow sufficient time for complexes to form. But it suggests only at room temperature, not 37C. In CRISPRMAXTM instruction, it also suggests that "Incubate for 5–10 minutes at room temperature", which Amila has already followed. So, I am not sure this (w/o pre-incubation at 37C) is really the problem!? 

I wonder whether or not this same approach will work on the plant protoplast cells (without cell wall).

Thanks for your input. It seems that other protocols use extended incubation times vs 5 mins with CRISPRMAX. This is also one possibility I will look into. 

About the tracrRNA, I'm using a 69mer. Its the same one as in the Aida paper I mentioned previously. Yuan-Yeu, thanks for sharing that paper. 

Based on your expertise, would you guys recommend me to use an even longer tracrRNA or switch to sgRNA? I'd like to hear your thoughts on this. Thanks!

Hi Amila,

Since you have all the materials now, I suggest that you should stick to your original plans for now and finish testing these things below first:

Testing:

1. pre-incubation at 37C

2. adjust the molar ratio of tracrRNA and crRNA

3. pre-incubation them at room temp for 15 min (extended time period)

Until all these variables are all tested and fail (hopefully not), then go for other options.

1. I read a couple of articles recently, including this one from Alt-R™ CRISPR-Cas9 System (see below)**.

• (a) It also mentions that 2-part crRNA:tracrRNA complex to guide and activate Cas9 outperforms other methods of delivering the CRISPR guide RNA components. (see below from Page 2 of the instruction).
• (b) The shortened tracrRNA seems perform very well and much better than the native one. But very important is that the tracrRNA has been chemically modified. This causes the major difference (of efficiency), and the major selling point of the kit. The chemically modified tracrRNA (and also crRNA) has a great ability to resist RNase (Page 3).

**From the instruction of Alt-R™ CRISPR-Cas9 System (on page 2):

Choosing the best CRISPR guide RNA format—sgRNA vs. separate crRNA and tracrRNA oligos:

It has been shown that the crRNA and tracrRNA can be fused into a chimeric single-guide RNA (sgRNA) [4,5]. This is convenient when expressing the guide RNA in target cells using a plasmid. However, because of their length, manufacturing sgRNAs as high quality RNA molecules by chemical synthesis is difficult and expensive. The sgRNA can be obtained by in vitro transcription, but this process is laborious, verification of sgRNA quality is difficult, and the resulting sgRNA can activate the innate immune system of many cells, causing cell death. Our data show that using a 2-part crRNA:tracrRNA complex to guide and activate Cas9 outperforms other methods of delivering the CRISPR guide RNA components.

2. I found another report researching on the efficiency of chemically modified sgRNA or 2-part crRNA:tracrRNA complex. I will post it as the next answer. (I don't want to post a long stretch of answer in the same answer area, which makes audience dizzy to read it.)

This is the article (from a Poster report) I mentioned in the previous answer.

In the poster, the authors reported that Chemically modified synthetic sgRNA or 2-part annealed synthetic crRNA:tracrRNA are clearly better than that of non-chemically modified counterparts. And the chemically modified sgRNA is superior in editing efficiency to a 2-part annealed synthetic crRNA:tracrRNA duplex.

Amila, has your crRNA or tracrRNA chemically modified? And, are you aiming for  knock-out or knock-in? (look like you just want knock-out result)

Yuan-Yeu, thank you for sharing very useful information. I will try another optimized transfection today and I'll let you know the results by the end of next week. 

Hi Amila,,

Have you gotten any results from the optimized transfection yet? Interested to know.

Hi Yuan-Yeu, 

The IDT method seems to have worked! However, I also see some cleavage in the control (no Cas9 or RNA, just GFP-plasmid), so that confused me at first and I thought the experiment failed. But after testing 5 different crRNA's I found that the cleaved bands are at the correct height, very different from control.

I will redo the analysis by using acrylamide gel, with the agarose gel the results are a bit faint but definitely there. 

I will post the results when I get them (I will be absent for two weeks). 

Thanks! Best, Amila

Hi Amila,

Thanks for your posting.

Could you direct me to the 'IDT method' site? Do they have a copy of PDF of the method available online?

What have you changed on the protocol? such as try pre-incubated them at 37C, which we discussed earlier?

Hi Yuan-Yeu, 

Sorry for my late reply, I've been absent for two weeks. I've got the protocol from the pdf you shared before (name: Alt-R CRISPR-Cas9 System) by IDT. See pages 10-13. I didn't change anything to the protocol and I followed it exactly. It seems to work well. 

Since sometimes the negative control looks false positive, I would suggest to try different sg/crRNAs to compare the different cleaved DNA products with the negative control.

Best, Amila

Thanks, Amila.

Amila Zuko It is quite common to see cleavage in the negative control of T7E1/surveyor assay due to existence of SNPs.

Dear Colleague,

I hope this message finds you well. Testing crRNA (CRISPR RNA) and tracrRNA (trans-activating CRISPR RNA) in vitro, particularly in cell culture, is an essential step in validating and optimizing CRISPR/Cas9-mediated genome editing. Below is a detailed and logical protocol that outlines the process of using crRNA and tracrRNA in cell culture.

Testing crRNA and tracrRNA in Cell Culture

Materials

Protocol

Troubleshooting Tips

• Transfection Efficiency: Optimize the transfection conditions for your specific cell line, including the amount of CRISPR components and transfection reagent.
• crRNA/tracrRNA Ratio: Ensure the crRNA and tracrRNA are annealed correctly and used in equimolar amounts.
• Cas9 Activity: Confirm the activity of the Cas9 protein by including a known target for editing.

Conclusion

Testing crRNA and tracrRNA in vitro in cell culture is a critical step in validating CRISPR/Cas9-mediated genome editing. By following the outlined protocol and incorporating appropriate controls, you can effectively assess the efficiency and specificity of your CRISPR system.

Perhaps this protocol list can give us more information to help solve the problem.