Dear Kunal,

Our lab has done genome editing by CRISPR/Cas9 in Xenopus tropicalis in the past few years and we have used several design tools so far, but in our experience and in our hands we have had the best results with the CRISPRScan tool from the Giraldez lab (PMID 26322839; http://www.crisprscan.org/). Additionally, it also immediately screens for off-target sites by two independent algorithms.

Designing sgRNAs with the CRISPRscan tool and injecting those with Cas9-NLS protein we routinely obtain around 50-95% genome editing effiency (targeted deep sequencing data-Miseq) with a succes rate of about 5/6 sgRNAs working from the first one we try.

On the topic of the design itself, if you use the "by gene" option of the CRISPRScan tool the tool will design them automatically against coding exons of the gene. Just make sure (via blatting in UCSC) that you do not target the last exon. We also target coding exons for genes and have by such method obtained good results.

For tropicalis researchers I can only wholeheartedly recommend to try CRISPRscan for the design of your sgRNAs.

good luck!

Thomas

Dear Kunal,

a very good platform for designing gRNAs is: http://crispor.tefor.net/crispor.cgi 

Here you could also check for off-targets in Xenopus species.

Hope that helps. Good luck for your experiments.

Best regards,

Bjoern

My labmates and I have had success using CRISPRdirect (https://crispr.dbcls.jp/). It can cross check the different xenopus genomes for tropicalis and laevis (at least what is deposited in Xenbase). Unfortunately, I have only designed them for knockin experiments by homologous recombination, so I cannot provide much input on the design for generating knockout animals. 

You can use this webtool : http://crispor.tefor.net/

a lot of species genomes are included for finding PAMs motifs.

If possible try to design your sgRNA only into the exonic sequences for making a KO. For making specific things (deletions, translocations,..) you may be using intronic sequences.

Dear Kunal,

Our lab has done genome editing by CRISPR/Cas9 in Xenopus tropicalis in the past few years and we have used several design tools so far, but in our experience and in our hands we have had the best results with the CRISPRScan tool from the Giraldez lab (PMID 26322839; http://www.crisprscan.org/). Additionally, it also immediately screens for off-target sites by two independent algorithms.

Designing sgRNAs with the CRISPRscan tool and injecting those with Cas9-NLS protein we routinely obtain around 50-95% genome editing effiency (targeted deep sequencing data-Miseq) with a succes rate of about 5/6 sgRNAs working from the first one we try.

On the topic of the design itself, if you use the "by gene" option of the CRISPRScan tool the tool will design them automatically against coding exons of the gene. Just make sure (via blatting in UCSC) that you do not target the last exon. We also target coding exons for genes and have by such method obtained good results.

For tropicalis researchers I can only wholeheartedly recommend to try CRISPRscan for the design of your sgRNAs.

good luck!

Thomas

hi, the CRISPOR homepage (see above) allows to screen for gRNA target sites for Xenopus tropicalis and laevis. It is my favorite site as it also has a scoring function for predicted on- target activity (6 different scores from different research groups are all integrated here). You can rank the results either in terms of specificity or related to the predicted on-target activity, which is great!

Hi Kunal, 

We have developed the tool CRISPRscan (Thanks a lot Thomas for recommend it!) and we are glad to see that is working pretty nice for zebrafish and also for Xenopus (our data and other people data). Selecting gRNAs with more that 55 will probably work good and with more than 70 will probably be excellent. We think that CRISPRscan is particularly useful for in vivo applications when you deliver gRNA and Cas9 (protein or mRNA). If you have any questions about how to use it, let me know and I will help you.

Best,

M.

The prokaryotic CRISPR (Clustered Regularly Interspaced Palindromic Repeats)-associated protein, Cas9, has been widely adopted as a tool for editing, imaging, and regulating eukaryotic genomes. However, our understanding of how to select single-guide RNAs (sgRNAs) that mediate efficient Cas9 activity is incomplete, as we lack insight into how chromatin impacts Cas9 targeting. To address this gap, we analyzed large-scale genetic screens performed in human cell lines using either nuclease-active or nuclease-dead Cas9 (dCas9). We observed that highly active sgRNAs for Cas9 and dCas9 were found almost exclusively in regions of low nucleosome occupancy. In vitro experiments demonstrated that nucleosomes in fact directly impede Cas9 binding and cleavage, while chromatin remodeling can restore Cas9 access. Our results reveal a critical role of eukaryotic chromatin in dictating the targeting specificity of this transplanted bacterial enzyme, and provide rules for selecting Cas9 target sites distinct from and complementary to those based on sequence properties.

http://elifesciences.org/content/5/e12677v2?_tmc=wX7DnKUXUWEPIrwLjJ7ou1pSeTp2b5ZJFxnKTwDUGHI

Wow!! Thank you everyone for your valuable inputs! Also, Miguel, congratulations on building CRISPRscan! I have found it extremely helpful. Also, thank you for your kind offer to help. :)

just one note: the scoring algorithm developed by Miguel is actually also integrated in the CRISPOR site and named Mor-Mateos!

Hello Dear Miguel, I have a question regarding gRNA design. Is it okay to select gRNA from last exon?? Pease guide me about gRNA selection from CRISPRScan. I am also using this site. It is really nice. What does CFD value actually mean?? Thanks in advnce.

Hi Ramendu, sorry for the very late reply, I did not see it till now!.

You can design gRNA whenever you like but causing out of frame mutations at the last exon will not generate loss of function mutants in your gene, most likely. I would recommend you to carefully read Vejnar, Moreno-Mateos et al., 2016 Cold Spring Harbor Protocols. There you will find a detailed protocol to design gRNAs. CFD is an score from Doench et al 2016, Nat biotech for off-targtes. Basically, the lower is the score the better the gRNA. Please Read Doench et al 2015 for more details.

Best,

M

Thank you so much@Miguel. Ihave designed gRNA according to the protocol you suggested, and I got very good results.