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  1. Evaluation Summary:

    Schubert and coworkers report the development of a novel CRISPR-based screening method that allows probing interactions between (a large set of) specific mutations and the abundance of specific proteins, and, more generally, investigate the spectrum of effects that (point) mutations can have on protein abundance. This complements existing strategies for measuring effects of genetic perturbations on transcript levels, which is important as for some proteins mRNA and protein levels do not correlate well. The ability to measure proteins directly therefore promises to close an important gap in our understanding of the links between genotype and phenotype, and the strategy is broadly applicable beyond the current study.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their name with the authors.)

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  2. Reviewer #1 (Public Review):

    Overall, the science is sound and interesting, and the results are clearly presented. However, the paper falls in-between describing a novel method and studying biology. As a consequence, it is a bit difficult to grasp the general flow, central story and focus point. The study does uncover several interesting phenomena, but none are really studied in much detail and the novel biological insight is therefore a bit limited and lost in the abundance of observations. Several interesting novel interactions are uncovered, in particular for the SPS sensor and GAPDH paralogs, but these are not followed up on in much detail. The same can be said for the more general observations, eg the fact that different types of mutations (missense vs nonsense) in different types of genes (essential vs non-essential, housekeeping vs. stress-regulated...) cause different effects.

    This is not to say that the paper has no merit - far from it even. But, in its current form, it is a bit chaotic. Maybe there is simply too much in the paper? To me, it would already help if the authors would explicitly state that the paper is a "methods" paper that describes a novel technique for studying the effects of mutations on protein abundance, and then goes on to demonstrate the possibilities of the technology by giving a few examples of the phenomena that can be studied. The discussion section ends in this way, but it may be helpful if this was moved to the end of the introduction.

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  3. Reviewer #2 (Public Review):

    Schubert et al. describe a new pooled screening strategy that combines protein abundance measurements of 11 proteins determined via FACS with genome-wide mutagenesis of stop codons and missense mutations (achieved via a base editor) in yeast. The method allows to identify genetic perturbations that affect steady state protein levels (vs transcript abundance), and in this way define regulators of protein abundance. The authors find that perturbation of essential genes more often alters protein abundance than of nonessential genes and proteins with core cellular functions more often decrease in abundance in response to genetic perturbations than stress proteins. Genes whose knockouts affected the level of several of the 11 proteins were enriched in protein biosynthetic processes while genes whose knockouts affected specific proteins were enriched for functions in transcriptional regulation. The authors also leverage the dataset to confirm known and identify new regulatory relationships, such as a link between the SDS amino acid sensor and the stress response gene Yhb1 or between Ras/PKA signalling and GAPDH isoenzymes Tdh1, 2, and 3. In addition, the paper contains a section on benchmarking of the base editor in yeast, where it has not been used before.

    Strengths and weaknesses of the paper:
    The authors establish the BE3 base editor as a screening tool in S. cerevisiae and very thoroughly benchmark its functionality for single edits and in different screening formats (fitness and FACS screening). This will be very beneficial for the yeast community.

    The strategy established here allows measuring the effect of genetic perturbations on protein abundances in highly complex libraries. This complements capabilities for measuring effects of genetic perturbations on transcript levels, which is important as for some proteins mRNA and protein levels do not correlate well. The ability to measure proteins directly therefore promises to close an important gap in determining all their regulatory inputs. The strategy is furthermore broadly applicable beyond the current study. All experimental procedures are very well described and plasmids and scripts are openly shared, maximizing utility for the community.

    There is a good balance between global analyses aimed at characterizing properties of the regulatory network and more detailed analyses of interesting new regulatory relationships. Some of the key conclusions are further supported by additional experimental evidence, which includes re-making specific mutations and confirming their effects on protein levels by mass spectrometry.

    The conclusions of the paper are mostly well supported, but I am missing some analyses on reproducibility and potential confounders and some of the data analysis steps should be clarified.

    The paper starts on the premise that measuring protein levels will identify regulators and regulatory principles that would not be found by measuring transcripts, but since the findings are not discussed in light of studies looking at mRNA levels it is unclear how the current study extends knowledge regarding the regulatory inputs of each protein.

    Specific comments regarding data analysis, reproducibility, confounders:
    The authors use the number of unique barcodes per guide RNA rather than barcode counts to determine fold-changes. For reliable fold changes the number of unique barcodes per gRNA should then ideally be in the 100s for each guide, is that the case? It would also be important to show the distribution of the number of barcodes per gRNA and their abundances determined from read counts. I could imagine that if the distribution of barcodes per gRNA or the abundance of these barcodes is highly skewed (particularly if there are many barcodes with only few reads) that could lead to spurious differences in unique barcode number between the high and low fluorescence pool. I imagine some skew is present as is normal in pooled library experiments. The fold-changes in the control pools could show whether spurious differences are a problem, but it is not clear to me if and how these controls are used in the protein screen.

    I like the idea of using an additional barcode (plasmid barcode) to distinguish between different cells with the same gRNA - this would directly allow to assess variability and serve as a sort of replicate within replicate. However, this information is not leveraged in the analysis. It would be nice to see an analysis of how well the different plasmid barcodes tagging the same gRNA agree (for fitness and protein abundance), to show how reproducible and reliable the findings are.

    From Fig 1 and previous research on base editors it is clear that mutation outcomes are often heterogeneous for the same gRNA and comprise a substantial fraction of wild-type alleles, alleles where only part of the Cs in the target window or where Cs outside the target window are edited, and non C-to-T edits. How does this reflect on the variability of phenotypic measurements, given that any barcode represents a genetically heterogeneous population of cells rather than a specific genotype? This would be important information for anyone planning to use the base editor in future.

    How common are additional mutations in the genome of these cells and could they confound the measured effects? I can think of several sources of additional mutations, such as off-target editing, edits outside the target window, or when 2 gRNA plasmids are present in the same cell (both target windows obtain edits). Could some of these events explain the discrepancy in phenotype for two gRNAs that should make the same mutation (Fig S4)? Even though BE3 has been described in mammalian cells, an off-target analysis would be desirable as there can be substantial differences in off-target behavior between cell types and organisms.

    In the protein screen normalization uses the total unique barcode counts. Does this efficiently correct for differences from sequencing (rather than total read counts or other methods)? It would be nice to see some replicate plots for the analysis of the fitness as well as the protein screen to be able to judge that.

    In the main text the authors mention very high agreement between gRNAs introducing the same mutation but this is only based on 20 or so gRNA pairs; for many more pairs that introduce the same mutation only one reaches significance, and the correlation in their effects is lower (Fig S4). It would be better to reflect this in the text directly rather than exclusively in the supplementary information.

    When the different gRNAs for a targeted gene are combined, instead of using an averaged measure of their effects the authors use the largest fold-change. This seems not ideal to me as it is sensitive to outliers (experimental error or background mutations present in that strain).

    Phenotyping is performed directly after editing, when the base editor is still present in the cells and could still interact with target sites. I could imagine this could lead to reduced levels of the proteins targeted for mutagenesis as it could act like a CRISPRi transcriptional roadblock. Could this enhance some of the effects or alter them in case of some missense mutations?

    I feel that the main text does not reflect the actual editing efficiency very well (the main numbers I noticed were 95% C to T conversion and 89% of these occurring in a specific window). More informative for interpreting the results would be to know what fraction of the alleles show an edit (vs wild-type) and how many show the 'complete' edit (as the authors assume 100% of the genotypes generated by a gRNA to be conversion of all Cs to Ts in the target window). It would be important to state in the main text how variable this is for different gRNAs and what the typical purity of editing outcomes is.

    Comments regarding findings:
    It would be nice to see a comparison of the results to the effects of ~1500 yeast gene knockouts on cellular transcriptomes ( This would show where the current study extends established knowledge regarding the regulatory inputs of each protein and highlight the importance of directly measuring protein levels. This would be particularly interesting for proteins whose abundance cannot be predicted well from mRNA abundance.

    The finding that genes that affect only one or two proteins are enriched for roles in transcriptional regulation could be a consequence of 'only' looking at 10 proteins rather than a globally valid conclusion. Particularly as the 10 proteins were selected for diverse functions that are subject to distinct regulatory cascades. ('only' because I appreciate this was a lot of work.)

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  4. Reviewer #3 (Public Review):

    This manuscript presents two main contributions. First, the authors modified a CRISPR base editing system for use in an important model organism: budding yeast. Second, they demonstrate the utility of this system by using it to conduct an extremely high throughput study the effects of mutation on protein abundance. This study confirms known protein regulatory relationships and detects several important new ones. It also reveals trends in the type of mutations that influence protein abundances. Overall, the findings are of high significance and the method appears to be extremely useful. I found the conclusions to be justified by the data.

    One potential weakness is that some of the methods are not described in main body of the paper, so the reader has to really dive into the methods section to understand particular aspects of the study, for example, how the fitness competition was conducted. Another potential weakness is the comparison of this study (of protein abundances) to previous studies (of transcript abundances) was a little cursory, and left some open questions. For example, is it remarkable that the mutations affecting protein abundance are predominantly in genes involved in translation rather than transcription, or is this an expected result of a study focusing on protein levels?

    Overall, the strengths of this study far outweigh these weaknesses. This manuscript represents a very large amount of work and demonstrates important new insights into protein regulatory networks.

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