View source for Design of guideRNA-Oligos (gRNA) for CRISPR/Cas gen. editing

{{Protokoll-Mix|Inhalt=
The genomic sequence (with exon/intron structure) of your target gene<br>Computer with internet-access to the Chop-Chop website (https://chopchop.rc.fas.harvard.edu/)<br>gRNA Oligos (sense/antisense) will be cloned in one of several vectors harboring a U6-promoter-driven RNA expression cassette<br>
Vectors used are:<br>pBluescript_U6-MCS (Plasmid #3468)<br>pBluescript_U6-MCS gRNA_Cerulean (Plasmid #xxx)<br>pX45xxx<br>pLenti}}

== Design principles for 20 bp gRNA ==

* Use the Chop-Chop website to find an appropriate gRNA target site upstream of a PAM sequence with no or low off-target probability 
* In general, try to target the first coding exon downstream of the initiating ATG start codon. Check that there is not an alternative ATG start codon in the proper reading frame nearby
* Make sure to use the correct genomic sequence (correct species) and target gene 
* in case of multiple splice variants, try to target a common exon

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<strong>Fig. 1: </strong>Exemplary scheme for the design of gRNA.
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