How to delete a short sequence with CRISPR/Cas9 on mESCs?

Hi Uwe Borgmeyer, thanks for your answer.

I have no other choice than directly targeting the site governing the 3 codons that I want to delete. If I were to target somewhere else, no matter if the recombination happens the enzyme will keep cutting the site unless an indel or frameshift takes place at the cut site. There must not be no single change in the sequence at the site other than the deleted 3 consequtive codons. So, if I were to target from within those codons -where I already have 2 target sites which are absolutely unique with their own PAMs- would it work if I only make use of a 100 nt long ssODN where those 3 codons will be deleted? How am I supposed to expect a strand invasion when those missing nucleotides will not anneal with my ssODN template(because they'll be missing in my template)? It seems complicated to me and I need some sort of illustration on how HDR with ssODN should work in this particular case. Thanks in advance.

Kumar Dilip

Yes ssODN based HDR should work, but make sure your ssODN has no extra mutations as long oligos synthesis always consists such abnormalities due to synthesis errors which you cant control. Best would be to use dsDNA template which you can control. HDR rate is low and your ssODN consists 10-20% errors that will make more challenging to get correct recombined clones at last

Uwe Borgmeyer

To get an overview I can recommend addgene.org (https://info.addgene.org/download-addgenes-ebook-crispr-101-2nd-edition). If you want to generate a mutated mouse line, starting with 1cell stage embryos would be easier than with stem cells. The advantage of ssODN is their high efficiency and low costs. You may consider a ssODN of 120bp which in your case lacks 9 nucleotides. It can displace one strand. Kumar is of cause right: You may get clones with additional mutations. However, compared to the InDels you get, they are very rare.

Onur Can Begentas

Thanks for your kind answers. Now I will try to clarify the case again with more details this time.

Let's suppose I use a protospacer targeting within those 9 nucleotides. How am I supposed to expect a recombination event where the ssODN template will not generate a proper 3'OH neither for hybridization (strand invasion) nor for polymerization -because my template will not have those missing nucleotides which I think will eventually occlude strand invasion-? The other issue is that, when we suppose I targeted outside those 9 nucleotides but still on the same exon, how can one expect the Cas9 activity to ever stop without introducing no single mutation to the protospacer after the cutting event?

So where do you recommend me to target in this case, and how to stop Cas9 activity after recombination in case I target outside of those 9 nucleotides? Because there should not be no more mutations other than a 9 nt deletion.

Uwe Borgmeyer

But it is the idea of using homologous recombination to alter a gene locus. You need homologous arms that flank new or mutated sequences in your case a deletion. It just works.

Concerning the other issue: Indeed Cas9 will cut again and it is very likely that you target the locus again which will result in InDels by non-homologous recombination. You may reduce this effect by using the protein and not an expression vector or an altered Cas9 with reduced half life (see e.g. PMID: 30517854).

Alternatively you may go for silent mutations or switch to an enzyme with another PAM sequence.

Kumar Dilip

Try to target pam within 9 nt if you find functional gRNA otherwise you have to mutate the pam in ssODN to avoid targeting by cas9. Before selecting gRNA check these things out. Select only those gRNA you could mutate their pam in the exon without changing the aa. In my knowledge ssODN will acts as a tamplate to be copied during repair. In that case repair will cover much more region than editing which allow to copy correct sequence from ssOD without 9nt

Yang Yu

Dear Colleague,

I hope this message finds you well. Deleting a short sequence using the CRISPR/Cas9 system in mouse embryonic stem cells (mESCs) involves designing and using guide RNAs (gRNAs) to target the flanking regions of the sequence to be deleted. Here is a detailed and logical protocol for achieving this.

Protocol for Deleting a Short Sequence in mESCs Using CRISPR/Cas9

Materials

mESCs: Cultured under appropriate conditions.

Plasmids:Plasmid encoding Cas9 nuclease (e.g., pSpCas9(BB)-2A-GFP, PX458). Plasmid encoding two gRNAs targeting the regions flanking the sequence to be deleted (can be in the same or separate plasmids).

Transfection Reagent: Suitable for mESCs (e.g., Lipofectamine 3000).

Antibiotic Selection: If using a selection marker (e.g., puromycin).

PCR and Sequencing Reagents: For validation.

Step-by-Step Protocol

Design gRNAs:Design two gRNAs that target sequences flanking the region to be deleted. Use online tools like CRISPR Design Tool, Benchling, or CHOPCHOP. Ensure the gRNAs have minimal off-target effects and are efficient in cutting the target site.

Construct gRNA Plasmids:Clone the gRNA sequences into a plasmid vector that also expresses Cas9. If using a dual gRNA vector, ensure both gRNAs are expressed from the same plasmid. Verify the sequences by Sanger sequencing.

Transfect mESCs:Seed mESCs in a 6-well plate at appropriate density (e.g., 2 x 10^5 cells per well) to achieve 70-80% confluency the next day. Prepare the transfection mix with the Cas9-gRNA plasmid and the transfection reagent according to the manufacturer's instructions. Add the transfection mix to the mESCs and incubate for 24-48 hours.

Selection (Optional):If using a selectable marker (e.g., GFP or antibiotic resistance), apply the selection to enrich for transfected cells. For example, add puromycin at an appropriate concentration to select for transfected cells.

Clonal Isolation:Single-cell cloning can be performed to isolate individual mESC clones. This can be done by limiting dilution or by picking individual colonies. Expand the isolated clones in a 24-well plate.

Screening for Deletions:Extract genomic DNA from the expanded clones using a standard genomic DNA extraction kit. Perform PCR using primers flanking the deletion site. Design primers that will produce different sized amplicons depending on the presence or absence of the target sequence. Run the PCR products on an agarose gel to identify clones with the expected deletion. Example PCR Setup: PCR Reaction Mix: 代码- Template DNA: 50-100 ng - Forward Primer: 0.5 µM - Reverse Primer: 0.5 µM - dNTPs: 200 µM each - PCR Buffer: as per manufacturer’s instructions - DNA Polymerase: 1 unit (e.g., Taq, Phusion) - Nuclease-free water: to final volume (25-50 µL) PCR Cycling Conditions: mathematica复制代码- Initial Denaturation: 95°C for 3 minutes - Denaturation: 95°C for 30 seconds - Annealing: 55-60°C for 30 seconds (optimize based on primer Tm) - Extension: 72°C for 1 minute per kb - Final Extension: 72°C for 5 minutes - Number of Cycles: 30-35

Sequencing Confirmation:Sequence the PCR products to confirm the deletion at the target site and to ensure that no unintended mutations were introduced.

Functional Validation:Perform additional assays to validate the functional impact of the deletion on the gene or regulatory element of interest.

Notes and Tips

gRNA Efficiency: Test the cutting efficiency of the gRNAs in vitro or in a pilot transfection to ensure high activity.
Off-Target Effects: Use high-fidelity Cas9 variants or engineered gRNAs to minimize off-target effects.
Clone Screening: Screen multiple clones to increase the likelihood of identifying successful deletions.

By following this protocol, you can effectively delete a short sequence in mESCs using the CRISPR/Cas9 system, ensuring precise genome editing and reliable experimental outcomes.

Should you have any further questions or require additional assistance, please feel free to reach out.

Reviewing the protocols listed here may offer further guidance in addressing this issue

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