Dear Anshika,

I have already used CRISPRs. But, I also consider same questions with you.

Since CRISPR causes double strand breaks by high efficiency in eukaryotic cells, you can chose any site for target sequence of gRNA, I think.

If anything, offtargets of the CRISPR are important .

Your question is how to reduce offtarget of CRISPR, right?

1. You have to chose unique sequences by some tool (Zifit or primer 3 or ....?).

2. You can use mutant Cas9 D10A, which is a nickase to cause single strand breaks.

Cas9 D10A is better to reduce the offterget of CRISPR, because a set of two Cas9D10A which bind close sites, is required to cause a double strand break.

Thus, the set of two Cas9D10A can induce mutations at a specific targeted site.

If you know more valuable information, please teach me.

Best,

Narumi Uno.

If you go to this website and follow the links for CRISPR, it will design the best gRNAs for your sequences.

http://zifit.partners.org/ZiFiT/

Hi here are some links;

http://www.e-crisp.org/E-CRISP/ (http://www.nature.com/nmeth/journal/v11/n2/full/nmeth.2812.html)

http://spot.colorado.edu/~slin/cas9.html

https://crispr.sigmainformatics.com/CrisprSearch.php

Good luck!

you could also use resources from Zhang lab/MIT

http://www.genome-engineering.org/

You can also use Off-Spotter, a very fast and exhaustive tool for CRISPR design, which our team created recently.

https://cm.jefferson.edu/Off-Spotter/

If you search for your gene, you will be able to see what gRNAs it contains, how many hits each gRNA has (including locations with 0-5 mismatches) and where exactly they are located on the genome. Plus you will get annotation info meaning you'll easily see what genes/transcripts they hit other than your gene.

Also you can visit wikipedia for a comparison between tools, https://en.wikipedia.org/wiki/CRISPR/Cas_Tools.

Hello,

Please can anyone tell me to what an extent mismatch in seed sequence is required to avoid offtarget.

Thanks

@Nishant.... I tried using benchling for designing Crispr gRNA for the gene of my interest. When i select the exon of interst for gRNA design , the results which are displayed shows +AAAGGGGTTTCCCC and right below that have another sequence -CCCCTTTAAAAGGGG

what does it mean ? which sequence should i select and what are next steps i have to follow?