Peer review of: Chromosome drives via CRISPR-Cas9 in yeast (2020)



Reviewers' Comments: 
 
Reviewer #2: 
Remarks to the Author: 
I am reviewer 2 from the initial set of reviews. The authors have overall made a good effort to 
improve the manuscript. With the following minor revisions, it should be suitable for publication in 
Nature Communications. 
 
Regarding the points of my previous review: 
 
1. The English language of the manuscript is substantially improved, but it still needs more work 
(I’m disappointed by the quality of the Nature editing service that the authors used - these issues 
should have caught by professional editors). Nevertheless, with the following additional revisions, 
it will be at an acceptable standard for publication. (In the following notation, the line listed is the 
starting line of the phrase to be corrected). 
Line 29: “transposon” should be “transposons” 
Line 44: “of Saccharomyces cerevisiae chromosome” should be “of a Saccharomyces cerevisiae 
chromosome”. 
Line 66: “exit” should be “exist”. 
Line 88: remove the word “And” (sentences cannot start with a conjunction) 
Line 136: “were driven by the violacein pathway” should be “inherited the violacein pathway” 
Line 137: “To verify chromosome elimination further,” should be “To further verify chromosome 
elimination,”. 
Line 187: “To confirm whether the strain containing chrXIV of Y12 has a thermotolerance trait 
further, using chromosome transfer and chromosome elimination,” should be “To further confirm 
whether the strain containing chrXIV of Y12 has a thermotolerance trait using chromosome 
transfer and elimination,” 
Line 249: remove the word “cause”. 
Line 252: should “HR” be “HDR”? 
Line 346: “1ml” should be “1 mL”. There are also other instances in the method where “l” should 
be capitalized and there should be a space between the quantity and the measurement unit. 
Line 566: “bolt” should be “blot” 
Line 574: “deviations” should be “deviation” 
Supplement Line 14: “bolt” should be “blot” 
Supplement Line 50: “and three of them displayed in the figure” should be “and three of them are 
displayed in the figure” 
Supplement Line 55: “Y12 behaved better thermotolerance compared to controls, indicating chrXIV 
of Y12 is one” should be “Y12 were more thermotolerant compared to controls, indicating chrXIV 
of Y12 is a” 
Supplement Line 68: “BY4741 in diploid strain” should be “BY4741 in a diploid strain” 
 
2-5. The revisions here are satisfactory. 
 
6. I’m glad this experiment was done, since the paper is now much more complete. However, it 
should still be noted by the authors (probably in the discussion) that recombination could still 
occur in the ~20% of cases (highly variable depending on the strain and target) where the target 
chromosome is not eliminated. This could separate the target site from the desired chromosome to 
be eliminated. This would not a problem for single-generation experiments or controlled crosses 
over multiple generations. However, it would be an issue in the classic gene drive experiment in 
which a few drive individuals are released into a non-drive population and expected to take over 
the population over the course of several generations. In this case, the final population would not 
consist entirely of the desired chromosome unless the chromosome elimination efficiency was 
extremely high (enough to prevent surviving chromosomes, which could recombine). Including this 
discussion would improve the paper. 
 

Minor points: All of these were suitably addressed. 
 
I have a few additional minor points for improving the clarity of the revised manuscript. 
 
1. Starting line 64, the authors write: “First, we designed a guide RNA (gRNA) targeting a specific 
sequence (5’- CATTATACGAAGTTATAAGTTGG-3’) that is 135 bp from the centromere of synIII, 
which does not exit in chromosome III (chrIII) of BY4741. For biosafety and efficiency, a molecular 
containment strategy was applied10.” 
This sort of reverses the logical order of things. The fact that the synIII target site exists on there 
IS the containment strategy. I suggest uses the following wording: 
“For biosafety and efficiency, a molecular containment strategy was applied10. We designed a 
guide RNA (gRNA) targeting a specific sequence (5’- CATTATACGAAGTTATAAGTTGG-3’) that is 135 
bp from the centromere of synIII, which does not exist in chromosome III (chrIII) of BY4741 or in 
wild-type yeast.” 
 
2. Within the supplement, the figures and tables should be labeled as “supplementary”. They 
should also be labeled in the order they appear in the text (for example, supplementary figure 5 is 
the first one referred to, so it should be designed as #1 and come at the beginning of the 
supplemental section of the manuscript). 
 
3. Supplement line 66 states that “Serial dilution assay analysis showed yHX0266 strains grew 

better than BY4741 under 40℃.” However. I think the authors meant AT 40 degrees, rather than 

UNDER 40 degrees (though they also seem better at 37 degrees). 
 
4. If possible, the panels in figure 3 should be arranged horizontally first and then vertically (so 
“B” should be where “D” is, “C” where “G” is, etc.). 
 
5. Check the formatting of the authors in reference 5. In general, the formatting of many 
references has minor issues (the usual ones from Mendeley - too much capitalization of titles, lack 
of appropriate italics, occasional spacing issues, etc.). 
 
6. The phrase “in an unbiased manner.” on line 247 is a bit unclear, since the main objective is to 
bias the inheritance. Consider removing it. 
 
7. Many of the semicolon breaks in the discussion should probably be completely separate 
sentences. 
 
8. On line 279 if this occurs, it definitely employs the same principle, not just “may”. The 
chromosome drive that the authors developed is certainly a form of CRISPR toxin-antidote gene 
drive in any case (with or without duplication). 
 
9. On line 292, it’s very unclear that chromosomal drive could be used in the treatment of 
diseases. End-joining is the dominant method in animals, and there is no sexual reproduction with 
somatic cells that would suffer from diseases. Consider removing this portion or elaborating on 
these factors. 
 
10. I have a minor quibble with the other reviewer regarding the definition of a “gene drive”. As 
noted in my original review, there are many toxin-antidote drives in existing (using both CRISPOR 
and other methods) that work not by directly increasing the number of gene drive alleles, but by 
removing wild-type alleles from the population via various methods (thus indirectly increasing the 
frequency of gene drive alleles in the population). I think that the author’s drive should therefore 
be called a “gene drive”, though describing it as a “meiotic drive” is also certainly accurate. On 
another note, the experiment where the components are episomally inserted would be considered 
a “split drive”. 
 

Figure 2A seems to be pretty strong evidence of endoreduplication taking place, since I don't think 
there is a better mechanism to get all four members of the tetrad to have EGFP. I'm assuming that 
the image is typical of tetrads from this cross. Figure 2E also seems to support this, indicating that 
all four spores were viable. 
 
Figure 2D based on sequencing depth could potentially be good evidence as well. My concern is 
that the authors may have just sequenced viable progeny, and of course, if a chromosome were 
lost, the individual would not be viable (thus preventing the "read depth" on ChrX from being 
reduced, even though at the tetrad stage, there would be half as many). Can the authors confirm 
that they were specifically sequencing tetrads here, which would ameliorate this issue? I wasn't 
clear on this based on the figure legend text, so they should probably revise the wording here.