How to design gRNA for CRISPR genome editing?

The gRNA guides the CRIPR Cas9 system to the right genomic location for performing a double strand break.The targerting specificity of the CRISPR Cas9 system is determined by the 20 nucleotides at the 5' end of the gRNA. The gRNA base pairs to the complementary  strand of the target sequence and Cas9  nuclease performs a  double strand break. 

While designing a gRNA following things must be taken into account.

1) GC content: Typical range is between 40% -  80% . A higher GC content stabilizes the RNA -DNA duplex while destabilisizing off-target hybridizations

2) Length: The length could be adjusted and range from 17-24 base pairs. A shorter sequence leads to minimized off target effects. ( 17 bp is the lowest limit on the length of the guiding sequence, any length of the guiding sequence any length shorter than 17 has a statistical chance of targeting multiple genomic loci.)

3)Potential off-target effects: Mismatch tolerance and the target site is what leads to off-target effects. This can be reduced by genome-wideoff target effect  searches.

There are many websites that help in designing gRNAs. Some of them are Chop Chop (Harvard), Broad Institute sgRNA designer, and Biotools.

I am explaining how to use Chop Chop website here,

1)  go to https://chopchop.cbu.uib.no/ 

2) Select Species

3) Select species specific gene id or cut and paste the nucleotide of interest, and start analysis.

Once analysis is done graphic representation of site specific gRNA will be shown. Each potential gRNA will also show the possible off target effects. Select the ones with less off target effects.