I am attempting to knock-out the gene F3'H in Arabidopsis (NC_003076.8). What is the recommended length of the gRNA? I understand that it should be 17-20nt long (+PAM), but which length will produce the best results?
According to the paper "Improving CRISPr-Cas nuclease specificity using truncated guide RNAs" (https://www.ncbi.nlm.nih.gov/pubmed/24463574) shorter (but not shorter than 17nt + PAM) gRNAs seems to be the best, but this paper is from 2014 and the researchers are using human cells, so I am not sure if their findings are relevant for plant cells in 2016.
Hi Sjur,
There is a knock-out line available: GK-629C11 (Appelhagen et al., 2014). It could save you some time if you are working with Col-0.
Best,
Boas
This paper is about minimizing the off-target affects and 17 nt is not a good idea (MIT guys found a modified Cas9 Protein to reduce the off-target effects in 2016). I presume you will use wt-CAS9 protein, you need NGG as PAM and 20 nt length of sgRNA. KO is simple with crispr, you will get really good yield. For large deletions I use 2 sgRNA flanked the coding region. To generate your guide RNA I would go for Chopchop (my favourite so far). goodluck
Hey,
My experience is 20bp length in the gRNaA seq gives best results. Especially if you are using the wt Cas9 system. All the best.
Ya totally agree with ozge, you need to use 20 nt length for sgRNA with PAM site. But of you want to have more nucleotide deletion you go by designing the multiple number of sgRNA flanked to the coding region.
Thank you all for your valuable answers, it's much appreciated. I have also been in contact with a technician working with CRISPR at Sigma that told me it's basicly impossible to know which gRNA-length will work the best, it all depends on a lot of circumstances. She recommended me to try several lengths if possible. I will follow your kind suggestions and start with 20nt.
Boas Pucker: My end goal is to knock out the same gene in Begonia, but because Begonia has not been sequenced yet I would like to try out the technique on Arabidopsis first to see if I can get the method to work.
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