Question: How to find which residues in a protein which are targetable by CRISPR
1
gravatar for abel.tan13
16 months ago by
abel.tan1310
abel.tan1310 wrote:

Hello,

I have some protein X with a known protein sequence which has both its cDNA and genomic sequence(incl. introns and UTRs) on genbank.

I have used random mutagenesis to create random mutations in the genomic sequences of protein X. I wish to use CRISPR Cas9 to target these mutations. However, Cas9 can only target codons which are up to 21 nt 5 prime from a GG (PAM) motif or 21 nt 3 prime from a CC motif in the genome. If I want to maximise specificity, then the range is only 11 nt 5 or 3 prime respectively. Hence I am aware that some residues`codons may be too far away from the PAM site to mutate using Cas9.

The problem is I dont have any idea how to determine which residues whose codons are too far away (greater than 21 or 11 nt) from the PAM motif! If the DNA sequence contains only cDNA, then I have some idea, but to complicate things the genomic sequences contains UTRs and introns, and they may contain PAM sites as well!

How should I go about trying to solve my problem?

Thanks for the Help!

crispr cas9 gene genome • 457 views
ADD COMMENTlink modified 16 months ago by genecats.ucsc560 • written 16 months ago by abel.tan1310

The problem is I dont have any idea how to determine which residues whose codons are too far away (greater than 21 or 11 nt) from the PAM motif!

You don't mention the organism, so my question is 'is the genome sequence not available for your organism?'

ADD REPLYlink written 16 months ago by theobroma221.1k

Its available . Its a human (H sapiens) Im also trying Mouse (mus musculus)

ADD REPLYlink written 16 months ago by abel.tan1310

So, shouldnt you use a genome browser to help answer your question?

ADD REPLYlink written 16 months ago by theobroma221.1k

Yes I did but searching manually for PAMs would be too time consuming. Is there any way I can use software to automate the problem (e.g find, blast etc)?

If I can get the locations of the CDS from the gDNA sequence then I can write a script to automate that for me. The problem is I dont know how to fetch the CDS regions from the gDNA from genbank or from the genome browser.

ADD REPLYlink written 16 months ago by abel.tan1310
0
gravatar for genecats.ucsc
16 months ago by
genecats.ucsc560
genecats.ucsc560 wrote:

You may be interested in the CRISPR Track at the UCSC Genome Browser. The track shows NGG PAM motifs within 200 bp of exons in a particular gene track (the gene track varies depending on the assembly). You can use the Table Browser to download data from this track in the region of your gene of interest and find the regions you are interested in.

If you are interested in finding CDS regions, you can use the Table Browser with your gene track of interest and select the chrom, cdsStart and cdsEnd columns. This previously answered mailing list question has some more information for you. However if you are looking for something more easily scriptable, you can use our public MySQL server genome-mysql.soe.ucsc.edu in order to download your data. Please note that usage of our public MySQL server is subject to a few restrictions so that you don't take the server down for all users.

If you have any more questions about using the UCSC Genome Browser don't hesitate to send an email to our mailing list: genome@soe.ucsc.edu. If your question includes private data you can send it to genome-www@soe.ucsc.edu instead.

ChrisL from the UCSC Genome Browser

ADD COMMENTlink written 16 months ago by genecats.ucsc560
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