I am trying to design the oligonucleotides used for making sgRNA plasmid used in CRISPR.The online tool that I used (https://chopchop.rc.fas.harvard.edu/) gives me the suggestion for choosing the sgRNA sequence.
However, the sequence is on the complementary strand of the DNA. Since the Cas9 will bind to the opposite strand of the pam sequence and cuts the DNA, I am confused how to choose the correct sequence.because the nucleotides to be added to each strand are different (below).
Suppose the PAM is AGG and the sgRNA sequence is: 5'-....GGCTAATTTCAGG-3' on the antisense DNA.
5'-ACACC-NNNNNNNNNNNNNNNNNNNN-G-3'
3'- G-NNNNNNNNNNNNNNNNNNNN-CAAAA-5'
What is the correct sequence to add instead of N? All the confusion is because the sequence is on the complementary sequence I guess.
Thanks for help!
Let Sigma help you design your gRNAs. I have attached an application. Once completed, send to [email protected]. The experienced technical service staff will design your gRNAs, show potential off-targets, and help you determine if plasmid is the most suitable format for your experiment. Plus, you can ask any question you would like along the way.
Hi Mohammad,
it is important to remember that the way you clone crRNA into your standard vector will position the 20bp sequence (without PAM) downstream of the U6 promoter and upstream of the "tracr" sequence. This is something that you should check in the final construct, so the cloning orientation and DNA strand can be easily deduced. Upon transcription the full lengths sgRNA will consist of cr+tracr. In this setup the crRNA is positioned as if the PAM was on the 3' end.
E.g. your target sequence:
5'-....GGCTAATTTCAGG-3'
the final plasmid should look like this:
5'-U6promoter-GGCTAATTTC-tracrRNA-3'
If necessary double check the sticky ends (highlighted below in italic) of the annealed oligos. As with standard cloning you can see where the ligation will happen and make sure that 20bp crRNA is upstream of tracr.
Zhang lab has useful protocols:
http://www.genome-engineering.org/crispr/?page_id=23
good luck in cloning,
Madina
The simple way I try to remember this is to always design my sequence so that the 3' end is directly before the NGG. So if my sequence is on the antisense strand and the sense strand reads " 5'-CCNATCGATC...-3' " then in order to have the 3' end of my sgRNA end directly before the PAM (NGG), I would want my the customized sequence of my sgRNA to end " 5'...GATCGAT-3' "
Designing single-guide RNA (sgRNA) from an antisense sequence involves a precise process to ensure specificity and efficacy for CRISPR-Cas9 mediated gene editing. Here’s how I typically approach the design:
1. Sequence Identification: I begin by identifying the target gene and obtaining its antisense sequence. The antisense strand is used as a template for sgRNA design since it is complementary to the sense strand that Cas9 will bind and cut.
2. PAM Sequence Location: I locate the Protospacer Adjacent Motif (PAM) sequence, which is essential for Cas9 binding. For Streptococcus pyogenes Cas9 (SpCas9), the PAM sequence is 'NGG'.
3. Protospacer Selection: I select a 20-nucleotide sequence immediately upstream of the PAM on the antisense strand. This forms the protospacer that dictates sgRNA specificity.
4. sgRNA Construction: I design the sgRNA by including the protospacer sequence and ensuring it is followed by the scaffold sequence necessary for Cas9 binding and DNA cleavage.
5. Off-Target Analysis: I use bioinformatics tools to analyze potential off-target effects by comparing the sgRNA sequence against the entire genome.
6. Synthesis and Verification: After designing the sgRNA, I synthesize it and verify the sequence via sequencing to confirm accuracy.
By following these steps, I design sgRNAs that direct Cas9 to the specific location on the DNA for targeted gene editing.
l Perhaps this protocol list can give us more information to help solve the problem.
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