Completed on 29 Apr 2016
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Reviewer 3 Advance Summary and Potential Significance to Field:
This manuscript describes an attempt to design high efficient guide RNAs for Crispr/Cas9 based mutagenesis in the ascidian Ciona. First, authors designed 83 guide RNAs that target genes expressed in the heart precursor cells in order to measure their mutation efficiencies. Based on this dataset, sequence preferences of good and not good guide RNAs were extracted. The information were then used to establish a program designing guide RNAs that are potent to have good mutation efficiencies in Ciona. Using the program authors made a list of recommended guide RNAs that covers almost entire region of Ciona genome. Crispr/Cas9 system provides us an easy method to knockout genes, and the simplicity of the method is good at genome wide analyses. Ciona is an excellent model for the genome wide analyses due to its small number of genes encoded in the genome. The program and the guide RNA sequences described here will be good resources to support future knockout analyses of this organism.
The experiments were done relatively thoroughly and extensively. Honestly speaking, I felt that the results are not so much novel, because the sequence preference of good sgRNA in Ciona generally confirms the previous data in other metazoans, and the gene functions described here are already known ones.
However, the presented algorism and Ci2KO dataset will greatly facilitate gene knockouts in Ciona and therefore this manuscript will be quite valuable for Ciona community. For non-Ciona researchers the methods presented here will be very helpful for constructing similar dataset and extracting the tendencies of good guide RNAs for the organisms. For these reasons I favor this manuscript for the techniques and resource section of Development. The text was written quite plainly enough for easy understanding.
Reviewer 3 Comments for the Author:
I found several relatively minor but essential flaws that need to be corrected or addressed before considering acceptance. I hope authors find my comments useful for improving the manuscript.
1. The authors initially evaluated the mutation efficiency of 83 guide RNAs.
How were these 83 sites chosen? I understand that most of the targeted genes are related to cardiovascular system. I would like to know whether there was any bias, or they were randomly selected, otherwise all were experimented?,
when picking up some from many potential target sites of a gene. If there is a bias, I would worry about the possibility that the bias might have influenced the following experiments such as extracting sequence preferences of guide RNAs.
2. Controls of FoxF expression. Authors showed the occurrence of large deletions of FoxF gene by simultaneously introducing two sgRNAs targeting FoxF. To show the bi-allelic deletions of the gene, in situ hybridization was carried out. As the control of the experiment, a sgRNA that is not related to FoxF was used. This is not a good control. Controls should be single FoxF sgRNAs- introduced embryos. The numbers of examined animals and FoxF-reduced animals were not shown (or I failed to find them). Adding them is necessary. In Figure 3, colonies 03, 04, 06 are indicated but I could not fully understand what does the information mean. If authors attempted to show the frequencies of large deletions, it is an important data and please explain more clearly.
3. I am interested in whether the mutation rates of many sgRNAs (the threshold of good one is 25%, or 37% if the underestimation is considered) can or cannot satisfy researchers, because I felt that mutation rates (or rates of phenotype appearance) in this study and related manuscripts would not always be so high enough to find novel phenotypes. Focusing on a specific phenomenon (like cardiovascular system in this manuscript), meaning that analyzing with expected phenotyeps, and using more efficient knockout/knockdown methods in parallel with Crispr/Cas9 would be a practical use of Ci2KO sgRNAs, and discussion like this kind may help readers' understanding. This comment was done because the manuscript focuses on the genome wide analyses: an important focus of this kind of studies is finding unexpected functions of genes. How about the mutation rate in other organisms in which F0-based analyses have been adopted? Some comparisons with the data of other animals would be useful too.
4. The statement of GC contents of human genome is wrong. Human genome is AT-rich like that of Ciona. References are necessary for the GC contents of Ciona and human.
5. I was surprised at the efficient electroporation of PCR product even though the amount of DNA is quite low. Is the linearization the cause? What does happen if plasmid DNAs are linearized before electroporation? The comparison may reveal why low amount PCRed DNA can express genes in high efficiency, and will be useful to improve electroporation, one of the greatest techniques in Ciona.