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

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
The authors outline a method to introduce episomal CRISPR components into yeast to enable 
biased inheritance of specific chromosomes. These systems include an episomal plasmid system 
capable of cleaving near the centromere, thereby preventing the inheritance of this chromosome in 
the progeny, and they demonstrate this is dependent on the proximity of the cleavage site with 
the centromere (Figure 1). They further use this system to demonstrate biased inheritance of a 
chromosome encoding genes required for the violetin pathway as a reporter. While they postulate 
this is due to a duplication of the Violitin-containing chromosome, we do not agree that this 
conclusively represents a duplication or if it is due to simple destruction and absence of the non-
engineered chromosome (Figure 2). In Figure 3 they outlined the modification of the system to 
enable biased inheritance of a set of species-specific ribosomal loci, and in Figure 4 they outline 
biased inheritance of a thermotolerant trait. In Figure 5 they use the system to elucidate location 
of a previously-uncharacterized trait of chromium resistance in an engineered SCRaMbLEd yeast 
system. 
 
Major corrections: 
- We understand this is likely an ESL manuscript. However throughout the manuscript there are 
significant grammatical mistakes, and improper use of scientific language that greatly hinders 
reader comprehension. We therefore recommend seeking proofing from a 3rd party, following such 
edits the intention of the authors will be much more clear. 
- Because the proposed system relies on destruction of the wild type chromosome (not the 
duplication or reproduction of the synthetic genetic element) should it be considered a true gene 
drive? Perhaps use of the term Meitoic drive would be more appropriate for this system, as it relies 
on destruction of the wt chromosome. However, in a traditional sense, drives are generally 
reserved for those expressing CRISPR components chromosomally, not necessarily episomally. Can 
the authors justify this design decision? 
- We would like to see a transgene map for the Cas9 and gRNAs throughout the work. 
- Line 30: Reference #4 is not necessarily what we would consider the primary, and most notable, 
literature outlining the biology of Selfish genetic elements. Can the authors justify their reasoning 
for selecting this specific reference for inclusion here? Or identify more relevant references to cite 
in its stead 
- The authors speculate that the entire chromosome is being driven (Figure 1). However, can 
recombination be ruled out? Are there markers on the engineered chromosome – distal to the 
CRISPR site – that could be assayed for to determine if the entire chromosome ‘drove’ as a single 
unit? 
- Figure 1: It would be nice to see an overview map of the CRISPR cassette itself. 
- Figure 1A. It would be nice if it were more clear that the orange chromosome was being 
destroyed in the figure. Currently, with the alignment between the grey and orange chromosomes, 
it appears as if they are oriented for homologous recombination – not destruction. 
- Figure 1B: A number of comments: The upper PCR gels in each panel (syn-PCRTag) lack a 
positive control. Most gels lack the required positive or negative controls throughout the work 
unfortunately. All gels should either include a ladder or a label of fragment size. The bands of 
these gels are sufficiently faint it is difficult to be sure they are truly PCR-positive as the authors 
suggest. And can the authors address why the band sizes are all different in these PCR fragments? 
- The authors should discuss the choice of Cas9 expression plasmid and its properties, if not in the 
results section, it would be nice to be given information on its properties in the methods section. 
The Methods section refer to Table 5 (which should also read “Supplementary Table 5” if it is to 
remain in the supplement), however Table 5 does not give detailed information on the plasmid 
properties. 
- Line 67: While the SynIII marker appears to be absent by PCR (assuming a positive control 
would have worked), this fact alone cannot prove that the entire chromosome has been ablated. If 
the authors were to PCR for other markers distal to the SynIII site, then this may be able to lend 

credence to their claims, however it would still not be sufficient for complete proof of the 
chromosome’s absence. The claims outlined here require further support. While it appears this is 
addressed in Figure 2, the aforementioned claims can not be supported by the findings in Figure 1 
alone. 
- Line 71-74: the genetics described is unclear here. 
- Line 110: How sure can we be that it has duplicated? Or is there just only one viable 
chromosome available, and the other mostly cleaved? Did they count the total number of tetrads 
that came out of the experiment? 
- With the CRISPR components expressed episomally, could this be considered a drive? If they 
were to insert the CRISPR components into the genome, it could be considered a selfish 
chromosome. 
- It would be a welcome addition for the authors to further elaborate on the practical application of 
such a system in the Discussion. Perhaps in industrial or agricultural applications, beyond mapping 
traits in a laboratory setting. 
- Line 230: I don’t know if they can make an association between their system and homology-
directed repair. I don’t believe they sufficiently demonstrated that homing was occurring, nor that 
the chromosome was duplicating (however I do believe their system is successfully preventing the 
cleaved chromosome from being inherited – however this is not the same as duplication). 
- 
 
 
Minor Corrections: 
- Line 11: add “The” in front of target chromosome and “the” before desired chromosome for 
proper use of English grammar. 
- Line 12: inclusion of the word “Whole” is considered inappropriate scientific language. 
- We understand this is likely an ESL manuscript. However there are significant grammatical 
mistakes, and improper use of scientific language throughout hindering reader comprehension. 
This review will therefore not seek to correcting for grammar, instead focusing the remainder of 
the review on Scientific merit and larger-scale errors under the assumption it will be proofed 
significantly prior to publication. 
- The sentence beginning in line 18 is a run-on. Please shorten for clarity. 
- Figure 1A: denote exact distance from centromere for precision (ie, not <200bp) 
- Line 28: specify that it is “normal Mendialian inheritance” for accuracy and specificity. 
- Line 33: reference 5 is incorrect 
- Line 34: References 6-9 are exclusively mosquito gene drive papers, but the authors mention 
yeast and mammals as well. They should add or modify the references cited to reflect this. 
- Line 40: It is not clear what the intended message of the sentence beginning with “It is still a 
challenge…” is. Please rephrase for clarity. 
- Line 63: the gRNA target sequence should include the 5’ and 3’ annotations. And the PAM should 
be distinguished in some way. This should be done for all mentions of gRNA target sequence and 
primers throughout the text. 
- Line 72: figure should be capitalized. 
 
 
 
Reviewer #2: 
Remarks to the Author: 
In this study, the authors design a new type of gene drive system in yeast called a chromosomal 
drive. Cas9 and gRNA cut a site near the centromere of a chromosome, resulting in destruction of 
the chromosome. The drive chromosome, however, does not contain the target site, thus biasing 
inheritance toward the drive. 
 
The toxin-antidote drive strategy was quite interesting. The paper will probably be impactful to the 
field and a more general audience. However, I do have some concerns that I believe must be 
addressed before the manuscript can be published. To give some perspective for this review, I 

specialize in gene drive but not in yeast genetics. 
 
 
Major Points 
 
1. The entire manuscript requires a thorough edit for proper language. This will be necessary to 
publish the manuscript in an English-language journal. To help, here is a version of the abstract 
that has been minimally revised to correct several English errors: 
“Self-propagating drive systems are capable of causing non-Mendelian inheritance and altering 
traits of populations. Here, we report a drive system called chromosome drive that eliminates a 
target chromosome and spreads a desired chromosome via CRISPR/Cas9. In our study, we found 
that a whole Saccharomyces cerevisiae chromosome can be eliminated efficiently through only one 
double strand break around the centromere using CRISPR/Cas9. As a proof-of-principle 
experiment of the CRISPR/Cas9 chromosome drives system, the synthetic yeast chromosome X 
was completely eliminated, and the counterpart wild-type chromosome X harboring a synthetic 
violetin pathway was driven throughout a population by sexual reproduction. We also demonstrate 
the use of chromosome drives to spread complex genetic traits in yeast. Megabase synthetic 
chromosome XII, heterogenous Y12 chromosome XIV, and SCRaMbLEd chromosome X were had 
biased transmission to progeny by chromosome drives in a population of Saccharomyces 
cerevisiae to spread a species identified ribosomal gene cluster from Saccharomyces bayanus, 
complex traits of thermotolerance, and unresolved traits of chromium resistance, respectively. 
Chromosome drives enables biased inheritance on a chromosomal scale, facilitating chromosome 
scale genetic mapping and extending applications of self-propagating drive.” 
 
2. Why does Figure 1B and 1C seem to imply 100% chromosome elimination, but then the 
experiments in Figure 3C indicate that elimination is usually 80-90% when the site is sufficiently 
close to the centromere? 
 
3. Why weren’t the yeast able to do end-joining repair for the Cas9-indced breaks. Is end-joining 
somehow inhibited near centromeres? If so, is this also true in other species? These matters 
should be included in the manuscript. 
 
4. The experimental details in Figure 2D are unclear, and I am unclear how to interpret the results 
of the spot picture. In general, I understand that the chromosome drive increased in frequency 
over three rounds or generations of mating, but I’m unclear what the starting and ending 
frequencies of the drive were, which is quite important for this sort of experiment. 
 
5. Can the authors comment why the deletion efficiency is so different between the different 
strains in Supplemental Table 2? Is the cut rate lower, or did DNA repair (HDR or end-joining) 
occur? This might require a quick sequencing experiment, but this is critical for understanding 
what is happening in these yeasts. 
 
6. Is there no recombination between chromosomes during yeast sexual reproduction? This could 
have an important effect on a chromosome drive and should be mentioned in the article if true 
(and discussed if not) to make it more accessible for non-specialist readers. 
 
 
Minor Points 
 
1. References 6 and 13 are the same. In general, in the introduction, it might also be useful to 
include several other CRISPR/Cas9 gene drives studies covering yeast and Drosophila (including a 
safeguarding study that used synthetic target sites). 
 
2. In the first results subsection, it would be useful to make it explicit that the WT chromosome 
does not have the gRNA target site. 

 
3. Figure 1B is redundant. It can probably be eliminated, since all the information is in 
Supplementary Figure 1. However, why was the band for the wt-PCRTag not present in a few 
samples? Also, “Left-M” and “T” should be defined int he legend for Figure 1D. 
 
4. Supplemental Table 1 would be better if it had percentages, while still specifying the sample 
size of 120. 
 
5. The idea of targeting an important site with CRISPR with a desired site is immune to cleavag4e 
has been explored as a gene drive in Drosophila. They drives themselves are quite different, but 
they share the same “toxin-antidote” principle. Indeed, the authors in the discussion say: “To 
achieve the purpose of biased inheritance of desired chromosomes by chromosome drives system, 
the process of chromosome endoreduplication may not be critical since elimination of target 
chromosome in the zygotes will result in inviability of progenies without desired chromosome.” 
This is the main method of previous CRISPR toxin-antidote drives and proposals. It might be worth 
including them in the introduction and/or discussion: 
https://www.nature.com/articles/s41467-020-14960-3 
https://bmcbiol.biomedcentral.com/articles/10.1186/s12915-020-0761-2 
https://www.biorxiv.org/content/10.1101/861435v2