I don't know that it could be done using lenti, as frameshift is unlikely to be sufficient. However, there have been publications with increasingly large sections of DNA removed from chromosomes and so creating a clonal cell line lacking the sequence would be quite feasible

Hi Lovorka

We used classical CRISPr to successfully deplete one lncRNA (double nicking system with a recombination donor vector harboring 2 flanking arms and a puromycin resistance gene in between). It works wonderful !. In fact I was thinking in using iCRISPR, but because of the low levels of expression of lncRNAs, I preferred to completely knock-down the gene.

Let me know if you need more details.

Best

Paco

Dear Lovorka

If I were you I would try to deplete lncRNA at RNA level because mostly they are in non-coding regions and it'll take a long time to have a comprehensive knowledge about this part of genome. For manipulating RNAs by CRISPR/CAS9 you should see Dr. Doudna's report " Programmable RNA recognition and cleavage by CRISPR/Cas9" where they introduce PAMmers to RCas9.

Good luck,

Hi again,

recent report from J. Weissman lab ( Cell 2013,) nicely reported depletion of several lcnRNAs by CRISPri. One could compare this method with introduction of 3xpoly A site and ask is it the RNA or active transcription that is important for lncRNA functions.

Hi Lovorka

This is different, since is a transcriptional inhibition by steric interference of an inactive Cas9 protein with the polymerase. However, a beautiful paper by Jeniffer Doudna in Nature showed how to use CRISPr to degrade directly RNAs, instead of DNAs. Take a look

http://www.nature.com/nature/journal/vaop/ncurrent/full/nature13769.html

You must choose what strategy is better for you.

Best.

Paco

to Francisco J. Enguita: How big was your deletion with the classical CRISPR?Thanks,Lo

Hi Lovorka, 

I have managed to delete 4 kb of lncRNA gene using Nickase version of Cas9 (with 2 pairs of guides on either side of a gene). I also use CRISPRi or CRISPR ON effectively on various lncRNAs in mouse ESC and MEFs.

let me know if you need more details

good luck

Thanks, that would be great. can I email you? Lo

My lincRNA is smaller... sorry... just around 1 kb...

Sure Lovorka, I am happy to receive emails 

Madapura Marulasiddappa Pradeepa : Hi, I am trying a similar strategy for deleting LincRNA in mESC's. Can you share the protocol that worked for you?. It will be very helpful for me. 

I have been talking to a representative from Sigma that suggested me to use two vectors expressing gRNAs designed at the beginning of the first exon and at the end of the last exon. After co-transfection of the two vectors (expressing also Cas9 of course) one can select for repair events that remove the entire lncRNA gene..... I'm consifering to give it a try. But maybe not with the vectors but with gRNAs and Cas9 transfection...

best

Ingram

Dear All,

I've just seen this thread. If anybody is interested in using CRISPR for lncRNA knockout, our recently developed DECKO vector system might be of help:

http://bmcgenomics.biomedcentral.com/articles/10.1186/s12864-015-2086-z

If you are interested, please drop me a line at the email address in the paper. We found CRISPR KO by paired guides to be pretty effective at knocking down lncRNA expression.

Best

Rory

Thanks Rory for sharing your paper, it is indeed a useful method for probing the function of regulatory elements. I will go through the paper soon. Are these plasmids deposited to Addgene? 

Hi

Plasmids are not in addgene just yet. If you get in touch through my work email indicated in the paper we'll be happy to share.

Best

Rory

Thanks Rory! I would be interested to try it and compare to CRISPRi which now we know that works. I will send you an email, thanks once again

Ok sure! we're very happy to share our method. 

Thanks Rory for sharing your paper!

Dear Lovorka, thanks for such question. I wonder if i could get the general protocol that worked for CRISPi in lncRNA and regards the method of transfection was lipofectamine or something else?

Gilbert et al., Cell 2014 is a good start when doing CRISPRi for lncRNAs.

Better to KD it at RNA level, see work from the Hsu lab OR by CRISPR/Ca9-mediated epigenetic silencing (but NO deletion).

Yes, CRISPR interference (CRISPRi) has indeed been applied to the study of long noncoding RNAs (lncRNAs). CRISPRi, which typically uses a deactivated Cas9 (dCas9) fused to a transcriptional repressor, allows for targeted silencing of gene expression, and it has been effectively utilized to investigate the functions of lncRNAs in various biological contexts. Here’s a detailed explanation of how CRISPRi is applied to lncRNAs:

CRISPRi has provided researchers with a powerful tool for studying the often elusive functions of lncRNAs, which are increasingly recognized for their roles in regulating gene expression, cellular differentiation, and disease pathology. By selectively repressing lncRNAs in a controlled manner, scientists can dissect their functional roles and potentially uncover new therapeutic targets.

With this protocol list, we might find more ways to solve this problem.