CRISPR/Cas9-Mediated Mutagenesis of GmFAD2-1A and/or GmFAD2-1B to Create High-Oleic-Acid Soybean
, Yupeng Cai 1,2,†, Qiang Su 1,2, Yingying Chen 1,2 and Wensheng Hou 1,2,*Round 1
Reviewer 1 Report (Previous Reviewer 1)
Here authors present an interesting biotechnological research on improving soybean oleic acid content using CRISPR-Cas knock-out of GmFAD2-1A and GmFAD2-1B. The manuscript is well written and structured. Authors imporoved the manuscript quality and have added to discussion a comparison of their results to the article with similar methodology and results, as I asked them in the first review. I recommend to accept this manuscript.
Author Response
Thank you very much.
Reviewer 2 Report (New Reviewer)
This paper describes the use of CRISPR/Cas9 technology to knock out two fatty acid desaturase genes in soybean to create breeding lines with high oleic acid percentage, and decreased percentage of polyunsaturated fatty acids in the seed. Overall, the manuscript is well-written, the methods are appropriately described, and results are properly presented with appropriate statistical differences indicated. I only have a few minor suggestions for improvement.
I suggest reworking the title to ‘CRISPR/Cas9-mediated mutagenesis of GmFAD2-1A and/or GmFAD2-1B to create high oleic acid soybean’
Put a / in between the two genes to indicate the double mutant: fad2-1a/fad2-1b
Section 2.4. How were the seed extracts prepared?
Check the figure captions. In Fig. 1, the caption says red lowercase letters indicate insertions, but there are no red lowercase letters. There is a blue one. In Fig. 2, it says that both insertions and base substitutions are indicated by red lowercase letters. Is one of these supposed to be blue? Also, explain what the blue capital letters indicate in Fig. 2.
Table 2. Please indicate the units (%) in the table caption and if the +/- is standard error or standard deviation.
Line 210. Define the abbreviation DAE (days after emergence) here, unless it is standard for this journal.
Some of the initials in the Author Contributions don’t match the author list. (S.S. for example)
Author Response
- I suggest reworking the title to ‘CRISPR/Cas9-mediated mutagenesis of GmFAD2-1A and/or GmFAD2-1B to create high oleic acid soybean’
Response: Thank you very much for your kind evaluation and insightful comments. We have changed the title according to your suggestion. It can be seen in lines 2-3.
- Put a / in between the two genes to indicate the double mutant: fad2-1a/fad2-1b
Response: Thank you very much for your kind evaluation and insightful comments. We have put a / in double mutant: fad2-1a/fad2-1b in the revised manuscript.
- Section 2.4. How were the seed extracts prepared?
Response: Thank you very much for your kind evaluation and insightful comments. We have added the seed extraction in the method. Heated methyl ester extraction method was used for fatty acid extraction. 20 soybean seeds were selected for each soybean line and grounded into fine powder with a grinding machine (RetschZM100, Φ=1.0mm, Rheinische, Germany). 0.03g soybean powder were placed in a 2 mL sterile centrifuge tube. 1 mL n-hexane were added into the centrifuge tube at 60°C for 20 minutes, shaking every 5 minutes. Then 1 mL sodium methanol solution (0.5 mol/L) was added into each centrifuge tube and oscillated for 10 minutes to completely methyl ester. Centrifugation at 13,000 rpm/min for 2 min, 200 μL supernatant was absorbed into a special sample bottle for chromatographic analysis. It can be seen in lines 103-110.
- Check the figure captions. In Fig. 1, the caption says red lowercase letters indicate insertions, but there are no red lowercase letters. There is a blue one. In Fig. 2, it says that both insertions and base substitutions are indicated by red lowercase letters. Is one of these supposed to be blue? Also, explain what the blue capital letters indicate in Fig. 2.
Response: Thank you very much for your kind evaluation and insightful comments. We have revised the figure captions. It can be seen in lines 159, 165.
- Table 2. Please indicate the units (%) in the table caption and if the +/- is standard error or standard deviation.
Response: Thank you very much for your kind evaluation and insightful comments. We have added the units (%) in the table caption and the +/- is standard error. It can be seen in lines 186, 188.
- Line 210. Define the abbreviation DAE (days after emergence) here, unless it is standard for this journal.
Response: Thank you very much for your kind evaluation and insightful comments. We have added the define of the abbreviation DAE. It can be seen in lines 215-216.
- Some of the initials in the Author Contributions don’t match the author list. (S.S. for example)
Response: Thank you very much for your kind evaluation and insightful comments. We have revised the Author Contributions. It can be seen in lines 279-281.
Reviewer 3 Report (New Reviewer)
The presented manuscript bring interesting and relevant information regarding high oleid acid in soybean. There are some aspects that should be improved:
Title: please rephrase, I do not think that expression “high oleic acid mutagenesis of ..”is a correct one
L23 there is no need for quotation marks for transgene-free
L23 in fact not only lacking Cas9 gene but whole T-DNA cassette?
L44 FAD2-1 italics
L46 GmFAD2-1 italics
L74-75 it was meant: Cas9 gene expression was driven by the CaMV 35S? (sgRNA expression too in L75, and bar gene L76)
L80 replace chosen with selected or designed using
L84 Agrobacterium tumefaciens italics (L85 also)
L86 why this cultivar?
L95-L96 what do you mean by analysis via sequence peaks?
L97 spaces missing
L99 bar italics
L105 more info regarding equipment
Fig 1 b what do you mean by sequencing the mutants? Please rephrase
L165 replace mutating with inactivation of
L185-L186 Cas9 gene instead Cas9 protein
L206 replace 2 with two
L208 replace on with in
Fig4 please include scales bars for pictures
Fig4 b, d and e: is it statistically different? Please note on the graph
Please give more information regarding the targeted sgRNA like exon region, potential off-targets.
Author Response
- Title: please rephrase, I do not think that expression “high oleic acid mutagenesis of ..”is a correct one
Response: Thank you very much for your kind evaluation and insightful comments. We have revised the title. It can be seen in lines 2-3.
- L23 there is no need for quotation marks for transgene-free
Response: Thank you very much for your kind evaluation and insightful comments. We have deleted the quotation marks. It can be seen in lines 22-23.
- L23 in fact not only lacking Cas9 gene but whole T-DNA cassette?
Response: Thank you very much for your kind evaluation and insightful comments. We have revised the sentence and deleted the lacking Cas9 gene. It can be seen in lines22-23.
- L44 FAD2-1 italics
Response: Thank you very much for your kind evaluation and insightful comments. We have revised. It can be seen in line 44.
- L46 GmFAD2-1 italics
Response: Thank you very much for your kind evaluation and insightful comments. We have revised. It can be seen in line 46.
- L74-75 it was meant: Cas9 gene expression was driven by the CaMV 35S? (sgRNA expression too in L75, and bar gene L76)
Response: Thank you very much for your kind evaluation and insightful comments. Both Cas9 gene and bar gene were driven by CaMV 35S. The sgRNA was driven by the Arabidopsis thaliana U6 promoter. We have revised the sentence. It can be seen in line 76.
- L80 replace chosen with selected or designed using
Response: Thank you very much for your kind evaluation and insightful comments. We have revised. It can be seen in lines 79-80.
- L84 Agrobacterium tumefaciens italics (L85 also)
Response: Thank you very much for your kind evaluation and insightful comments. We have revised. It can be seen in lines 84-85.
- L86 why this cultivar?
Response: Thank you very much for your kind evaluation and insightful comments. The cultivar Jack has a high transformation rate.
- L95-L96 what do you mean by analysis via sequence peaks?
Response: Thank you very much for your kind evaluation and insightful comments. The heterozygous mutations showed overlapping peaks from the target sites to the end. The wild-type and homozygous mutations had no overlapping peaks at the target sites. Then, the homozygous mutant types were identified by sequence alignment with the wild-type sequence.
- L97 spaces missing
Response: Thank you very much for your kind evaluation and insightful comments. Tsingke is a Biotechnology Company’s name.There is no space in Tsingke.
- L99 bar italics
Response: Thank you very much for your kind evaluation and insightful comments. We have revised. It can be seen in line 97.
- L105 more info regarding equipment
Response: Thank you very much for your kind evaluation and insightful comments. We have added the produced company about the equipment. It can be seen in lines 111-112.
- Fig 1 b what do you mean by sequencing the mutants? Please rephrase
Response: Thank you very much for your kind evaluation and insightful comments. The heterozygous mutations showed overlapping peaks from the target sites to the end. The wild-type and homozygous mutations had no overlapping peaks at the target sites. The sequencing can be showed the mutant types.
- L165 replace mutating with inactivation of
Response: Thank you very much for your kind evaluation and insightful comments. We have revised. It can be seen in line 170.
- L185-L186 Cas9 gene instead Cas9 protein
Response: Thank you very much for your kind evaluation and insightful comments. We have revised. It can be seen in line 190, 195.
- L206 replace 2 with two
Response: Thank you very much for your kind evaluation and insightful comments. We have revised. It can be seen in line 211.
- L208 replace on with in
Response: Thank you very much for your kind evaluation and insightful comments. We have revised. It can be seen in line 213.
- Fig4 please include scales bars for pictures
Response: Thank you very much for your kind evaluation and insightful comments. We have revised the Fig.4. The scales bars were added. It can be seen in Fig. 4.
- Fig4 b, d and e: is it statistically different? Please note on the graph
Response: Thank you very much for your kind evaluation and insightful comments. The statistical method is same. The results showed no significant in germination rate, flowering time and maturity time. We have noted on the graph in Fig.4. It can be seen in Fig. 4.
- Please give more information regarding the targeted sgRNA like exon region, potential off-targets.
Response: Thank you very much for your kind evaluation and insightful comments. The sgRNA information is in lines 130-132.
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
This work is an interesting biotechnological research on improving soybean oleic acid content using CRISPR-Cas knock-out of GmFAD2-1A and GmFAD2-1B. The manuscript is well written and structured, results are clearly presented. That said, there is an article with similar methodology and results as in the manuscript (Do, P.T.; Nguyen, C.X.; Bui, H.T.; Tran, L.T.N.; Stacey, G.; Gillman, J.D.; Zhang, Z.J.; Stacey, M.G. Demonstration of Highly 289 Efficient Dual Grna Crispr/Cas9 Editing of the Homeologous Gmfad2-1a and Gmfad2-1b Genes to Yield a High Oleic, Low 290 Linoleic and Alpha-Linolenic Acid Phenotype in Soybean. BMC Plant Biology 2019, 19, 311.). It is cited in the introduction, but not discussed in the discussion section of the manuscript. I think it would improve overall quality of the paper and confirm novelty and significance of the results if you add a comparison with that published work to the discussion section.
Reviewer 2 Report
In the submitted manuscript “Improved Soybean Oleic Acid Content by CRISPR/Cas9 Targeted Mutagenesis of GmFAD2-1A and GmFAD2-1B” by Fu et al., the authors generated GmFAD2-1A and GmFAD2-1B-edited mutants using CRISPR/Cas9-mediated gene editing technology and the homozygous mutants showed dramatic increases in oleic acid content. However, it is worth noting that an almost identical approach with results has been published by another group in 2019 [Do, et al. "Demonstration of highly efficient dual gRNA CRISPR/Cas9 editing of the homeologous GmFAD2–1A and GmFAD2–1B genes to yield a high oleic, low linoleic and α-linolenic acid phenotype in soybean." BMC plant biology 19.1 (2019): 1-14]. Therefore, I do not recommend this manuscript for publishing.
