Cas13 sgRNA design

[Aaron]

Hi Genome Engineers,

I was wondering if there is any existing tools for sgRNA design for Cas13a or Cas13b. 

Kindly let me know.

best,

Aaron 

[nan...@mail.dfci.harvard.edu]

I have the same question. 

[Bhavana Muralidharan]

HI

I want to design sgRNA to clone in the vector provided by Zhang lab on addgene for cas13. I have some queries regarding the same. Are there any tools to do so. Or is anyone ready to discuss. My query is whether the oligo to be cloned should have the same sequence as the mRNA ( i.e. the 5' oligo should have the same sequence as the mRNA or should it be reverse complement.

Thanks

Bhavana

[Omar Abudayyeh]

Hi all,

Jonathan and I in the Zhang lab are currently developing an online tool for his. Please let us know if you have any specific feature requests and we'll try to incorporate them!

Best,

Omar

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[Jin]

Hi Bhavana

I also plan to design the crRNAs and have the same question. Did u get the answers? If so, could you please let me know.

Thanks,

Jin 

[doubl...@gmail.com]

Hi Omar,

I am currently planing to use PspCas13b system for gene knockdown. I searched the plasmids on addgene, and found that cas13b vector is lentivirus but the crRNA vector are not. I might need the lentivirus system for my cells. So do you have the lentivirus plasmid for crRNA in your lab? or one lentivirus plasmid containing both cas13b and crRNA, just like the lentiCRISPR v2 plasmid. 

Thanks a lot.

Xiao

在 2018年2月5日星期一 UTC-6下午9:30:05，Omar Abudayyeh写道：

[CB]

any updates?

[Florian U]

Any news on the online tool? 

Or more generally: What guidelines - if any - would you suggest for PspCas13b guide design? In Cox et al., it is stated that "...a PFS constraint for interference in mammalian cells likely does not exist...". I read this like I can basically target whereever in the mRNA of my interest and expect robust knockdown, right?

[Jonathan Gootenberg]

Hi Florian,

We should have a demo of the tool in the next month or two. In the meantime, you're correct in that the PspCas13b guides don't have any particular constraints -- I would design 3 or so crRNA per gene that you want to target.

Let us know if you have any other questions!

Best,

Jonathan

On Tue, Apr 17, 2018 at 11:20 AM Florian U <7ull...@gmail.com> wrote:

Any news on the online tool? 

Or more generally: What guidelines - if any - would you suggest for PspCas13b guide design? In Cox et al., it is stated that "...a PFS constraint for interference in mammalian cells likely does not exist...". I read this like I can basically target whereever in the mRNA of my interest and expect robust knockdown, right?

--

[Kevin Du]

Hi, Omar,

It seems that the crRNA is no difference between Cas13a and Cas13b. Am I correct?

Kevin.

[adambr...@gmail.com]

Hi Omar, Jonathan and all Zhang lab members, 

Just wondering if there are any updates regarding tools for PspCas13b guide design by any chance please?

Continue being awesome!

Cheers, 

Adam

[Hadi Bayat]

Hello,

you may find the following online tool useful for Cas13a:

http://bioinfolab.miamioh.edu/CRISPR-RT/interface/C2c2.php

[Kangxin He]

Dear Bayat, is this site work well?

I just have found this website, hoping it helps.

btw, Dear everyone, how many guys are developing the cas13-based diagnositc tools for pathogens, same as me?

[Hadi Bayat]

It seems working aptly. But there is a limitation! it is optimized only for Cas13a and as it was elucidated, Cas13a is less efficient in eukaryote! 

Dr. Zhang et al. reported Cas13b is more efficient than Cas13a. Previously, Dr. Gootenberg mentioned an online tool will be established soon. 

[yhirao...@gmail.com]

Dear Bayat,

Do you have any idea about this site with compare to other siRNA design tools?

Which one is better to design effective crRNA?

[Hadi Bayat]

Dear Yuichi Hiraoka

unfortunately I don't compare this site with other siRNA design tools. However, it is previously indicated that Cas13a and Cas13b have  similar sequence constraints and sensitivities against mismatches in mammalian cells (in fact, there is no PFS constraint for interference)  and  are highly specific compared to a position-matched shRNA. Please see the paper entitled "  RNA editing with CRISPR-Cas13" published by Cox et al. (2017). So, it can be concluded that CRISPR-RT (http://bioinfolab.miamioh.edu/CRISPR-RT/interface/C2c2.php) currently is able to be applied for knockdown experiments with Cas13b. Moreover, Dr. Abudayyeh and Dr. Gootenberg mentioned "they are developing an online tool for Cas13b and it will be introduced soon".

Best,

Hadi

[Yuichi Hiraoka]

Dear Bayat

Thanks for your kind reply.
And happy to hear that CRISPR-RT is able to be applied for Cas13b.
Recently I prepareed several crRNAs desighned by different tools.

Maybe I will be able to update my result about comparision of each tools.

Ofcource I'm waiting for the tools for Cas13b.

Many thanks,

Yuichi

2019年1月13日日曜日 19時36分14秒 UTC+9 Hadi Bayat:

[Ishola Afeez]

Dear Bayat,

 

Your post was really helpful in designing crRNA for my proposed LwCas13a RNA editing experiment  but I am encountering serious issue concerning cloning the crRNA. I already saw the crRNA cloning vector from addgene (pC016 - LwCas13a guide expression backbone with U6 promoter)  but I am  not sure which position to insert the crRNA sequence. Meanwhile, let me mention that I am new to CRISPR/Cas system experiments. In addition, I also read that one can synthesis the crRNA. Please, your kind assistance in form of advice will be greatly appreciated. Hoping for your kind response. Thank you so much.

 

Best Regards,

Ishola Afeez  

 

On Wednesday, November 28, 2018 at 4:08:06 AM UTC+8, Hadi Bayat wrote:

[Hadi Bayat]

Dear Ishola

Hello,

unfortunately, I do not work with your desired plasmid. But it seems like other plasmids introduced by Dr. Zhang's lab, you should insert your gRNA into BbsI restriction site. Just be careful about obligate sticky ends that must be added to your ordered oligos. please kindly see the attached files.

All the best,

[Maheswaran Kesavan]

Dear Jonathan,

Is there any updates on this demo tool.

I saw in CHOP CHOP software that there is an additional option for RNA editing. I want to design gRNA for RNA targeting. Is there any tool which I can use ?

Thank you.

On Friday, May 11, 2018 at 4:06:55 PM UTC+1, Jonathan Gootenberg wrote:

Hi Florian,

We should have a demo of the tool in the next month or two. In the meantime, you're correct in that the PspCas13b guides don't have any particular constraints -- I would design 3 or so crRNA per gene that you want to target.

Let us know if you have any other questions!

Best,

Jonathan

On Tue, Apr 17, 2018 at 11:20 AM Florian U <7ull...@gmail.com> wrote:

Any news on the online tool? 

Or more generally: What guidelines - if any - would you suggest for PspCas13b guide design? In Cox et al., it is stated that "...a PFS constraint for interference in mammalian cells likely does not exist...". I read this like I can basically target whereever in the mRNA of my interest and expect robust knockdown, right?

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[Paula Coutinho]

Any updates on gRNA design using Cas13? :)

[James Mann]

  I've developed an automated Primer + gRNA / crRNA  pair generation tool for diagnostic approaches. I have a manuscript planned for Bioinformatics. Email me (James...@Baylor.edu)  if any of you are interested in oligo + crRNA generation. 

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