CRISPR/Cas9 Preferred method

Hi,
In C. elegans animal model, I am trying to tag an essential gene at the C’ with “GFP_SEC_Cre” using Cas9 RNP based CRISPR, and has no luck yet! I have tried using both (i) plasmid DNA (As described by Goldstein lab; https://doi.org/10.1534/genetics.115.178335), and (ii) dsDNA donor (as described by Mello lab; https://doi.org/10.1534/genetics.120.303564) as a repair template (individually), but no success yet. I haven’t find any rollers (sqt-1 inserton based)!

I would like to have suggestions regarding preferred Cas9 and sgRNA sources, i.e. plasmid DNA or Protein and sgRNAs from IDT company? Also, any other tips are much appreciated.
Thank you so much!

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Hi there,

So far I’ve used CRISPR/Cas9 only to delete regions but it should work the same for insertions when using a repair template.

I am using purified Cas9 protein from IDT. The tracrRNA and crRNA are also from IDT and i design the crRNA using their tool. ssODN can also be ordered from IDT and as far as I understood there seems to be a higher recombination efficiency when using ss over ds.

What is really important with Cas9 is the concentration ratio.

Here is what I use:

  1. 1.5ul tracrRNA 100 um + 2.2ul crRNA 100um heat at 95°C for 5min, then 25° for 5min
  2. add 1ul of 2.5ug/ul Cas9 to the above mix and keep at 25° for 5min
  3. then add 125ng total selection marker plasmid and
  4. 1 to 2 ul ssODN 100um
    IDT Buffer up to 10ul total volume

All this is centrifuged for 15min at 13000rpm and then injected without touching the pellet site. I always make the mix fresh in the morning of the injection and then keep it at room temperature afterwards until the injection time. So far I have always had transformants.

Hope it helps!

All the best,
Maria

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Thank you Maria! I will try this way.

SEC is too big to integrate using the RNP CRISPR method, which drops off dramatically in efficiency as the insert size increases. If you want to use SEC you should use a plasmid based method as described in the SEC papers. Also note the Jorgensen lab has deposited new Cas9 expressing worms at the CGC. I have had good luck using one of these (EG9882) with SEC inserts and 400-700 bp homology arms.

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We’ve used both methods frequently. A few questions:

  • how many animals are you injecting for the SEC and RNP approaches?

  • what is the crRNA/sgRNA target sequence?

  • for the RNP approach, how far from the cut site is the insertion, and what side of the PAM is it on?

One possibility is that you have the bad luck of having an inefficient guide. Barth’s suggestion about the Jorgensen stable Cas9 is good. If you check out that pre-print (Fig 1) you’ll see that occasionally one will get guides that fail and one needs to try a new guide (https://www.biorxiv.org/content/10.1101/2021.08.03.454883v1.full.pdf)

One way to test your guide is to inject the RNP, pool 10 marker positive animals, Sanger sequence them and run them through the free ICE tool on the Synthego website (h/t Julian Ceron-Madrigal for that tip). It will let you evaluate indel formation by Cas9 to see if you are getting cutting.

One other suggestion is to try different amounts of Cas9 protein (and even try the glh-1 locus as a control if you haven’t had GFP knock-ins with RNPs before). With our Cas9 prep, we needed to use a higher concentration than those that the Mello lab uses to get efficient editing.

Good luck!

Jordan

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Thanks for the reply, and sorry for replying lately.

Thank you Jordan for the reply, and sorry for the super delayed response!
I have beed injecting ~20-25 worms, and the two cut sites I tried are with in 30-40 bp (at C’); one cuts on the right side and other on the left since the respective crRNA are in Fwd & Rev directions.

One of the KI worked for me, with RNP recipe, only using N2 strain but not other like JIM113. I am wondering if the efficiency also varies with the strain!
I have to try higher concentration of Cas9, but even with the concentration mentioned in Mello lab recipe we are having Needle clogging issues.! So, would you share your recipe?

Many thanks again!

Tushar.

Thank you Maria for sharing your recipe! And sorry for replying super late.