Hi Moria, 

I don't have any suggests about changing that particular codon, however you could use the Cas9 nickase approach instead of the fully active Cas9. If you used the Cas9 nickase, you'd need to transfect your cells with two different sgRNAs (one targeting the forward DNA strand, one targeting the reverse DNA strand), which would mean you could look into changing the NGG sequence at the other Cas9 target site in your donor plasmid. If you find that you can change the other PAM sequence in your donor vector, that would solve your problem because it would prevent cleavage at that DNA locus, therefore a double strand break would not be formed and mutagenesis would not occur after successful DNA repair using your donor plasmid. 

It may be that you can't find two suitable Cas9 target sites close enough together in your gene of interest to use the Cas9 nickase approach, but it is worth looking into as an idea. This system also reduced the chances of Cas9 off target effects, as it requires two single strand breaks to be formed on opposite DNA strands in close proximity for the cell to recognise it as a DNA double strand break, instead of immediately forming a double strand break with a single enzyme. Plasmids I have used in the past for this sort of experiment are pX459 or pX461 (you will find these and many more available on Addgene). 

Best of luck! 

Jonathan 

Thanks Jonathan!

That`s a great idea. I`ll simply change the guide sequence in my donor plasmid, thus avoiding re-recognition of the entire cutting site by the cas9.

Thanks again!

Moria

Instead of mutating the PAM you could also introduce several silent mutations within the seed region of the sgRNA adjacent to the PAM. Two to three silent mutations may do the trick and inhibit efficient sgRNA binding. Also, if you are doing a knock-in, depending on your repair template, size and location of the insert, you may have already split the sgRNA target sequence in half or something by inserting your knock-in. So modification of PAM is not always needed.

I agree with Jonathan, also apart from this you can also opt for CRISPR/CPF1 based editing system. It uses TTTN as PAM site rather than NGG for Ca9. Also it creates staggered ends with are highly suitable for Knock-in as well..

Best Wishes 

Modifying the NGG protospacer adjacent motif (PAM) sequence in your donor plasmid for CRISPR applications without altering the amino acid sequence of the protein of interest, particularly when the amino acid is proline, requires careful consideration of the genetic code and potential strategies for synonymous codon usage.

Proline is coded by the codons CCU, CCC, CCA, and CCG. If the NGG sequence, which is essential for Cas9 recognition and binding, overlaps with a proline codon in your target sequence and you wish to change it without altering the proline residue, you will have to consider the following strategies:

1. Synonymous Codon Substitution

If the PAM sequence overlaps with a proline codon, you can look at substituting the codon with another that codes for proline but does not include the NGG sequence. For instance, if the sequence is CCG (Proline) with the G being part of the NGG PAM sequence, you could substitute it with CCU, CCC, or CCA, assuming this does not inadvertently create another PAM site or affect the expression and function of the protein.

2. Avoiding the PAM Sequence in the Donor

For homology-directed repair (HDR) applications, where the donor plasmid is used to introduce a specific change or insert at the CRISPR-Cas9 cut site, ensure that the PAM sequence modification is outside of the homology arms or in a region that does not interfere with the integration of the donor DNA. This way, the necessary sequence for Cas9 recognition and cutting is preserved in the genomic DNA but not necessarily present in the repaired or inserted sequence.

3. Designing Around the PAM

If modifying the PAM sequence could affect the expression or function of your gene of interest, consider redesigning your CRISPR strategy. This might involve choosing a different target site altogether that allows for efficient CRISPR-Cas9 editing without requiring modification of critical or sensitive sequences.

4. Utilizing Different Cas Variants

Explore using Cas9 variants or other CRISPR-associated (Cas) enzymes that recognize different PAM sequences. For example, SaCas9 recognizes NNGRRT sequences, and Cpf1 (Cas12a) recognizes TTTV sequences. By selecting an enzyme with a PAM requirement that doesn't overlap with your sequence of interest, you might avoid the need to modify the PAM sequence in your donor plasmid.

5. Silent Mutation Strategy

For cases where a synonymous codon substitution strategy is employed, it's crucial to verify that the silent mutation does not affect the mRNA's secondary structure, stability, or the protein's folding and function. This can be evaluated using bioinformatic tools and subsequent experimental validation.

6. Consulting with Experts

If unsure, consulting with a molecular biologist or a genetic engineer experienced in CRISPR-Cas9 genome editing could provide additional insights or alternative strategies tailored to your specific project needs.

In conclusion, altering the NGG PAM sequence in a donor plasmid for CRISPR without changing the amino acid sequence involves strategic use of synonymous codons, careful planning of the CRISPR edit to avoid critical sequences, and potentially employing different Cas enzymes to accommodate your genomic editing needs.

l With this protocol list, we might find more ways to solve this problem.