Donor design for multiple gRNA in C-terminal gene tagging

[francesco forin]

Hello dear CRISPR/Cas9 people,

my name is Francesco and I want to use CRISPR/Cas9 to incorporate a tag at the C-terminus of different genes (GFP, m-Cherry, FLAG, His).

I already have successfully design and used gRNAs for Knock-out of these genes so I am quite confident how CRISPR/cas9 works in general but when it comes to HDR Knock-in I have some doubts.

If I have a PAM sequence that allows me to cut right before the stop codon (ex: 5'-NNN LAST EXONE||TAGCGGNNNN-3') the design of the donor is quite simple: 5'- |NNN + LAST EXONE|--|TAG + STOP|--|NNNNNNN| -3'

                                                                                                                                                                                                                                                                                 (LEFT HA)                                       (RIGHT HA)

1) If the PAM sequence allows a cut let's say 5 bp after the stop codon in the 3'-UTR, does the design of my donor change? If yes, how?

2) If it does not change, can I use the same donor with different gRNAs (each one cutting at different positions, like 5, 10, 13 bp after the the stop codon).

Thank you in advance to everyone for you time and help! :)

Best,

Francesco

[Bruce Conklin]

Something else to think  about.  Since you have been knocking out genes, you are used to selecting guides with high NHEJ damage.  In contrast, with tagging you want to select for guides that make double stranded breaks followed with perfect repair.  This means guides with the "best" T7 assay may be the "worst" for making clean insertions.

[Bruce Conklin]

In gene knockouts, "active guides" are defined by high NHEJ damage.  For precise genome editing, what you want is cleavage with perfect repair.  These sites are "silently repaired" and not seen in a T7 assay.  I would screen by HDR, or Excision for cleavage activity. Then I would screen by T7 to get rid of the damaging cut sites.  Silent repair, is an ideal path to precise editing.

[Joe Miano]

Hello Francesco,

Your question is a good one.  Assuming you are using wtCas9 and not any of the growing number of gene editing endonucleases (eg, Cas12a, re-engineered Cas9 with diff PAM specificity) you will want your DSB as close to the stop codon as possible.  You are probably okay with using the same donor repair template with cut sites up to 15-20 nt away from stop, but you may have issues with incorporating the tag with sgRNAs that are much further away since repair is not via conventional HR, but involves SSTR etc,

We routinely tag genes in the mouse and we use a hybrid forward primer (comprising 5'endogenous gene..tag..3') and a reverse primer/  There's much in way of mosaicism but this can be easily screened against with judiciously designed primers (see Suppl Fig VI  in the attached, somewhat outdated, review of ours).

One last point: it should be possible to multiplex and target multiple loci simultaneously or the same locus with different tags that you can sort out upon breeding the mice.

Good luck!
Joe