length of gRNA

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regulatingge...@gmail.com

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Oct 19, 2016, 4:47:08 PM10/19/16
to Genome Engineering using CRISPR/Cas Systems
Hi,
Does the gRNA have to be 20 nucleotides. Somebody suggested using 19 nucleotides can also work but I wanted to check here on the forum first?
Thanks.

Ramesh Chandra

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Oct 19, 2016, 10:54:51 PM10/19/16
to Genome Engineering using CRISPR/Cas Systems
In my opinion 20bp better but 19bp also fine.

Thanks,

Ramesh

S Verkuijl

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Oct 20, 2016, 10:30:07 AM10/20/16
to Genome Engineering using CRISPR/Cas Systems

Truncated gRNAs (protospacer <20 nucleotides) can decrease undesired mutagenesis at off-target sites in general without sacrificing on-target genome editing efficiencies. 
If that is something that is crucial to you application it might be worth considering. 

Fu Y, Sander JD, Reyon D, Cascio VM, Joung JK.
Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat. Biotechnol.

Sjur Sandgrind

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Oct 20, 2016, 11:51:10 AM10/20/16
to Genome Engineering using CRISPR/Cas Systems
I am working on plant transformations and asked Sigma the same question a few weeks ago. They answered "We recommend 19 bp (without PAM) because in our research we have determined this to be an optimal balance between specificity and efficiency."
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