Online cas9 Designer and Off-target Explorer

[dayong guo]

Dear all,

Recommend an online tool made by myself:

cas9.wicp.net

It is a combination of 

1) high throughput auto design of multiple cas9 targets with fewer off-targets, labelled as features in a genbank file;

      and

2) thorough analysis of all possible off-targets in a genome, with coordinates and scores.

so, you can 

• Design cas9 in batch and avoid off-targets before it happens;
  
• Locate and eliminate off-targets after it's been done.
  

Any suggestion, pleas email me: guod...@gmail.com

Thanks to the Zhang lab who made the wonderful cas9 vectors and this discussion forum!

Day

[VajraDog]

Hi Dayong,

Thanks for providing this. I've a question, when you scan for off-target in mouse genome, does your algorithm use C57/Bl6? I'm asking this because my sequence is showing 1 mismatch on the 20bp, and ignoring the NGG sequence (which shouldn't be the case, it's a perfect match for B6). Thanks in advance.

Vajra

[dayong guo]

Vajra,

Thanks for your question. Your comment is important and I will add the database instruction to cas9.wicp.net ASAP.

The mouse genome db i used is a soft-masked file downloaded from the following link:

ftp://ftp.ensembl.org/pub/release-74/fasta/mus_musculus/dna/

with instruction of this:

http://www.ensembl.org/Mus_musculus/Info/Annotation#assembly

As you can see from ENSEMBL instruction:

"The primary assembly, GRCm38, was released by the Genome Reference Consortium in January 2012. It is based on Mus musculus strain C57BL/6J. This assembly is used by UCSC to create their mm10 database."

Wish this is helpful.

Day

[Fatwa Adikusuma]

Cool tool. I like it because it can predict the off target random sequences that do not exist in WT sequence (like bowtie). Could you please add human database as well? Thanks.

Fatwa 

[dayong guo]

Cannot forget homo, ourself.  will do for sure. :-)

[VajraDog]

thanks Dayong -Vajra

[dayong guo]

Dear all,

Thanks to your suggestions. Currently, mouse, rat, human, zebrafish, fruitfly genomes are all included in the COD (cas9 and off-target designer) system (http://cas9.wicp.net/)

(Please Note: the offtarget score is NOT just a score from BLAST search. you can consider it as the likelyhood or activity of a potential off-target, given the real target score as 1.)

dayong

[dayong guo]

Both NGG and NAG are considered when exploring off-targets.

Dayong

[DONGSHAN YANG]

Hi, Dayong,

 

Great tools! Is it possible to add rabbit and pig genomes to the species?

 

Thank you!

 

Dongshan 

[dayong guo]

Sure, will add pig, rabbit, and a kind of fish in early Feb 2014. Currently, i m on vacation without access to the server.

thanks for your suggestion!

Day

[Viet Dung]

I had intended to study the application CRISPR gene function in a crustacean. I expected to be able to design guide vector bearing specific promoter from this species. But I'm missing the gene sequence encoding NLS fused dCAS9 and hCAD9 for constructing CAS9 vector. I have no funds to buy CRISPR/CAS9 vector at this time, I hope some of you could share me a vector that contain CAS9 fused NLS gene for free. Vector can be easily sent by drop solution of plasmid on whatman paper and sent it by mail. I am in Gent, Belgium. I really appreciate the care and help of all of you. My email vietd...@gmail.com.

Viet Dung

[dayong guo]

Dear all,

The cas9 design system, COD, has included pig, rabbit, stickleback genomes, as requested by users.

Also, recent updates made off-target counting more acurate and handy.

After a month of my vacation, I found COD has generated 1000 results for users all over the world! Thank you for your support.

Day

Kill to Heal

COD is a hobby website built to acknowledge the sacrificed research animals and plants. 

As a biologist who knocks-out genes and kills creatures, you can heal, as well.

On Monday, December 23, 2013 4:03:15 PM UTC+8, dayong guo wrote:

[Karim Roder]

Hi Day,

that's great. Many thanks. Howevere, when I anted to try out my rabbit sequence I realized that the website is not working.

Cheers,

karim

[dayong guo]

Hi there,

Rice and Cow genomes included as requested. 

Thanks.

Day

On Monday, December 23, 2013 4:03:15 PM UTC+8, dayong guo wrote:

[dayong guo]

Hi there,

Rice and Cow genomes included as requested. 

Thanks.

Day

On Monday, December 23, 2013 4:03:15 PM UTC+8, dayong guo wrote:

[Jens Christian Eriksen]

Many thanks Day! Awesome tool!

[dayong guo]

hi, dear there,

there have been several improvements recently on the COD system ():

• mouse, rat, zebrafish off-target result tables have been modified to access -+300bp genome sequence directly, easy to design PCR and sequencing primers.
• CHO (Chinese hamster ovary) cell genome included. Pharm company users could be happy with it.
• Short gRNA of 17~20 nt can be labelled as features in cas9 design outcome, corresponding to recent publication saying shorter gRNA could be more specific. Short gRNA of 17~20 nt can be adapted in off-target estimation.
• Dictyostelium genome included.

Any suggestion, please let me know.

Day
On Monday, December 23, 2013 4:03:15 PM UTC+8, dayong guo wrote:

[dayong guo]

Update sum of COD (cas9.wicp.net) till Feb-28-2014:

• Version Feb-28-2014 (beta) Adjusted options of considering "NGG" or "NGG and NAG" as PAM when counting off-targets.
• Version Feb-14-2014 (beta) mouse, rat, zebrafish and human off-target result tables have been modified to access -+300bp genome sequence directly.
• Version Feb-11-2014 (beta) CHO (Chinese hamster ovary) cell genome included.
• Version Feb-10-2014 (beta) Short gRNA of 17~20 nt can be labelled as features in cas9 design outcome.
• Version Feb-8-2014 (beta) Dictyostelium genome included; Short gRNA of 17~20 nt can be adapted in off-target estimation.
• Version Feb-7-2014 (beta) Rice and cow genomes included.
• Version Feb-4-2014 (beta) Pig, rabbit, stickleback genomes are loaded; Off-target score can be selected by user (0, 0.05, or 0.25).
• Version Feb-3-2014 (beta) More off-targets included by an increased E-value. Off-target scores are limited to >0.05.
• Version Jan-2-2014 (beta) both NGG and NAG considered in off-target analysis, assuming the NAG as a PAM of lower activity than NGG.
• Version Dec-25-2013 (beta) Fruitfly (Drosophila melanogaster) genome database added.
• Version Dec-24-2013 (beta) Human genome database added.
• Version Dec-21-2013 (beta) New website with CSS.
• Version Dec-20-2013 (beta) Off-target scoring system added.
• Version Dec-13-2013 (beta) Off-target stringency added.
• Version Dec-10-2013 (beta) Zebrafish genome database included.
• Version Dec-9-2013 (beta) Public beta test version cas9.wicp.net started.
• Version Dec-7-2013 (beta) Modified off-target algorithm to be more precise.
• Version Dec-5-2013 (beta) Beginning of the beta test (internal).

Currently adding chicken genome as requested.

Thanks for all your suggestions!

Day

On Monday, December 23, 2013 4:03:15 PM UTC+8, dayong guo wrote:

[dayong guo]

Dear All, 

Thanks for your suggestions and participation. More species have been included as below:

• Version Mar-5-2014 (beta) Arabidopsis, Pseudomonas putida and Physcomitrella patens genomes have been added as requested.
• Version Mar-3-2014 (beta) Off-target scoring matrix modified according to two publications.
• Version Feb-28-2014 (beta) Adjusted options of considering "NGG" or "NGG and NAG" as PAM when counting off-targets. Chicken genome added as requested. Chicken off-target table have been modified to access -+300bp genome sequence directly.

• Version Feb-14-2014 (beta) mouse, rat, zebrafish and human off-target result tables have been modified to access -+300bp genome sequence directly.
• Version Feb-11-2014 (beta) CHO (Chinese hamster ovary) cell genome included.
• Version Feb-10-2014 (beta) Short gRNA of 17~20 nt can be labelled as features in cas9 design outcome.
• Version Feb-8-2014 (beta) Dictyostelium genome included; Short gRNA of 17~20 nt can be adapted in off-target estimation.
• Version Feb-7-2014 (beta) Rice and cow genomes included.
• Version Feb-4-2014 (beta) Pig, rabbit, stickleback genomes are loaded; Off-target score can be selected by user (0, 0.05, or 0.25).
• Version Feb-3-2014 (beta) More off-targets included by an increased E-value. Off-target scores are limited to >0.05.
• Version Jan-2-2014 (beta) both NGG and NAG considered in off-target analysis, assuming the NAG as a PAM of lower activity than NGG.
• Version Dec-25-2013 (beta) Fruitfly (Drosophila melanogaster) genome database added.
• Version Dec-24-2013 (beta) Human genome database added.
• Version Dec-21-2013 (beta) New website with CSS.
• Version Dec-20-2013 (beta) Off-target scoring system added.
• Version Dec-13-2013 (beta) Off-target stringency added.
• Version Dec-10-2013 (beta) Zebrafish genome database included.
• Version Dec-9-2013 (beta) Public beta test version cas9.wicp.net started.
• Version Dec-7-2013 (beta) Modified off-target algorithm to be more precise.
• Version Dec-5-2013 (beta) Beginning of the beta test (internal).

Have a nice day!

Day

On Monday, December 23, 2013 4:03:15 PM UTC+8, dayong guo wrote:

[CRISPY-AUS]

is it only me who is experiencing problem with this site http://cas9.wicp.net. First it let  me enter my 20 nt target sequence from mice and and the displayed the message- safari can not find the page. When I tried again, it didn't even let me put the sequence.

Cheers

[dayong guo]

Hi, CRISPY-AUS,

Sorry about that. Must have been the moment I updated the server to add more species, and links to the UCSC genome map viewer.

It should be fine now with new functions. Have fun!

Day

[dayong guo]

Today, the COD (cas9.wicp.net) system has processed its No.2112 sequence requested from all over the world! Thank you for your suggestions in the past two months.

Day

On Monday, December 23, 2013 4:03:15 PM UTC+8, dayong guo wrote:

[dayong guo]

Dear friends,

E. coli genome has been included in COD (cas9.wicp.net). It is the No. 18th species loaded in the system as listed: http://cas9.wicp.net/link.html

Thanks for your suggestions. Thanks to Dr. Feng Zhang's forum as a share point for everyone.

A High-throughput gene Knockout pipeline will be released recently. You can snap your genes of interest with cas9 in seconds. All you need to do is type in your gene ID!

Day

On Monday, December 23, 2013 4:03:15 PM UTC+8, dayong guo wrote:

[meke...@gmail.com]

It would be great if you could include the sheep genome as well! 

Thanks,

Kelsey

[dayong guo]

Hi, dear all,

COD (http://cas9.wicp.net/) will be offline for 4 days starting from tomorrow, then back online next week.

Sorry for the interruption.

Day

On Monday, December 23, 2013 4:03:15 PM UTC+8, dayong guo wrote:

[Chenghua Li]

Hi Dayong and all,

    I was told that 30bp guide should be used for pX260 or pX334. But your tool can only output <20bp gRNAs. Can anyone tell me how to solve this problem?

    Thanks.

Chenghua

在 2013年12月23日星期一UTC-5上午3时03分15秒，dayong guo写道：

[Ancheto]

Hi Dayong,

Is this tool still alive?

Thanks,

Ancheto

[Najla Arshad]

Hello!

I used this tool in October 2014, but now when I go back to it I get an error as below: 

No data received

Unable to load the webpage because the server sent no data.

Error code: ERR_EMPTY_RESPONSE

Is there some maintenance going on? I hope the site is not down!! 

Thanks,

Najla

[Najla Arshad]

Hello again!

I was trying to access the site:  : http://cas9.wicp.net/  but am unable to. I want to include this in the M&M of my paper so was trying to access the site to know how to cite it. Is it still live and may I know the best way to cite it?    

Thanks!