Rapid and specific degradation of endogenous proteins in mouse models using auxin-inducible degrons
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Evaluation Summary:
This manuscript will be of interest to the broad class of biologists and especially mouse geneticists who study the function of protein-coding genes. The authors confirm the utility of the auxin-inducible degron tool to rapidly degrade the target protein of interest by developing genetically modified mouse models. This expands the set of tools to study gene function in a cell/tissue type, in adults (bypassing embryonic lethality) and also to more finely dissect the different functions of pleiotropic genes.
(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, Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)
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Abstract
Auxin-inducible degrons are a chemical genetic tool for targeted protein degradation and are widely used to study protein function in cultured mammalian cells. Here, we develop CRISPR-engineered mouse lines that enable rapid and highly specific degradation of tagged endogenous proteins in vivo. Most but not all cell types are competent for degradation. By combining ligand titrations with genetic crosses to generate animals with different allelic combinations, we show that degradation kinetics depend upon the dose of the tagged protein, ligand, and the E3 ligase substrate receptor TIR1. Rapid degradation of condensin I and II – two essential regulators of mitotic chromosome structure – revealed that both complexes are individually required for cell division in precursor lymphocytes, but not in their differentiated peripheral lymphocyte derivatives. This generalisable approach provides unprecedented temporal control over the dose of endogenous proteins in mouse models, with implications for studying essential biological pathways and modelling drug activity in mammalian tissues.
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Evaluation Summary:
This manuscript will be of interest to the broad class of biologists and especially mouse geneticists who study the function of protein-coding genes. The authors confirm the utility of the auxin-inducible degron tool to rapidly degrade the target protein of interest by developing genetically modified mouse models. This expands the set of tools to study gene function in a cell/tissue type, in adults (bypassing embryonic lethality) and also to more finely dissect the different functions of pleiotropic genes.
(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, Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)
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Reviewer #3 (Public Review):
Genetically engineered mouse (GEM) models containing either recombinases (Cre and Flp) or drug inducible platforms (such as tetracycline inducible system) have been widely used for conditional and inducible control of gene inactivation. These systems achieve control of gene expression at the genetic level and these strategies achieve inactivation of the target protein after a considerable amount of time (usually several hours to days). However, there are not many examples of tools to achieve the same at the protein level and to quickly inactivate proteins. In this manuscript "Rapid and specific degradation of endogenous proteins in mouse models using 2 auxin-inducible degrons", the authors demonstrate the application of a plant-based chemical degradation system called auxin-inducible degron (AID) to …
Reviewer #3 (Public Review):
Genetically engineered mouse (GEM) models containing either recombinases (Cre and Flp) or drug inducible platforms (such as tetracycline inducible system) have been widely used for conditional and inducible control of gene inactivation. These systems achieve control of gene expression at the genetic level and these strategies achieve inactivation of the target protein after a considerable amount of time (usually several hours to days). However, there are not many examples of tools to achieve the same at the protein level and to quickly inactivate proteins. In this manuscript "Rapid and specific degradation of endogenous proteins in mouse models using 2 auxin-inducible degrons", the authors demonstrate the application of a plant-based chemical degradation system called auxin-inducible degron (AID) to inactivate target proteins, as quickly as 1 hour.
The degron system, by far, has been used in cell culture models. Its limitations such as leakiness and the requirement of higher doses of the substrate [indole-3-acetic acid (IAA)] and the toxicity associated with such high doses had diminished the enthusiasm of researchers in developing GEM models to test the system. The pioneering inventors of the degron system (Masato T. Kanemaki's group) recently made improvements to the AID, termed AID2, which overcame these limitations, and thus they could demonstrate the application of the degron mediated degradation of target proteins by generating GEM models. Very recently, another group (preprint Abuhansem et al 2021; now published in Developmental Cell, April 2022) successfully generated degron GEM models. These studies are set as initial examples of successful use of the degron system in mouse models.
Although not novel, the authors in this manuscript elegantly show the utility of the degron system. The authors developed degron GEM models for two proteins condensin I and condensin II and performed a series of experiments using primary lymphocytes isolated from the models and by using the animal models themselves. The experiments are well designed, the data agree with the interpretations and the conclusions are logical.
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Reviewer #2 (Public Review):
The authors of this study have successfully generated a sophisticated novel system to alter the degradation of specific proteins using a heterologous system derived from plants. They used the auxin-inducible degron (AID) approach by constructing and validating, in a robust stepwise manner, the different compounds for this ternary system to operate. First, by tagging the selected proteins (NCAPH and NCAPH2, two compounds of condensin chromosomal complexes) with AID and a fluorescent reporter protein, using CRISPR tools. Second, by creating a single copy-inserted transgene expressing TIR1, the E3 ligase substrate receptor, targeted into the Rosa26 locus and under the control of the CAG promoter. And third, by exposing cells or animals to the ligand, the plant hormone auxin, and studying the kinetics and dosage …
Reviewer #2 (Public Review):
The authors of this study have successfully generated a sophisticated novel system to alter the degradation of specific proteins using a heterologous system derived from plants. They used the auxin-inducible degron (AID) approach by constructing and validating, in a robust stepwise manner, the different compounds for this ternary system to operate. First, by tagging the selected proteins (NCAPH and NCAPH2, two compounds of condensin chromosomal complexes) with AID and a fluorescent reporter protein, using CRISPR tools. Second, by creating a single copy-inserted transgene expressing TIR1, the E3 ligase substrate receptor, targeted into the Rosa26 locus and under the control of the CAG promoter. And third, by exposing cells or animals to the ligand, the plant hormone auxin, and studying the kinetics and dosage of all three compounds. The authors took good care to document that the indicated genetic alterations did not significantly interfere with normal physiology. Although some overt phenotypes were reported (reduced size and fertility) they correctly concluded their system was not fundamentally impairing endogenous cellular processes. Next, upon recreating the double mutant mice, they went in a stepwise manner, to explore the efficacy of their approach first in several primary cell types and, later, directly in vivo, both in adult stages and during embryo development. Their findings convincingly demonstrate their ability to target the selected proteins for degradation and the associated alterations (such as mitotic arrest) that were not universally found, suggesting organ- and cell-specific limitations yet to be defined, possibly related to the capacity of TIR1 to function in all cell types or to the actual arrival of auxins to all cellular destinations. Their experimental system is used to document that some cell types appear to be dependent on these condensin complexes to complete their cell cycles whereas other cells appear to complete the mitotic cycle in their absence, suggesting additional mechanisms of mitotic control to be further studied. Even with the limitations expressed by the authors themselves, this novel approach to assessing protein function appears to be highly powerful and might be applied to a variety of biological questions, now directly targeting proteins, beyond previous studies whose rationale was limited by the inducible and conditional gene expression, where DNA (and not proteins) was targeted hoping to foresee phenotypes associated with subsequent protein synthesis alteration. In summary, a technically brilliant study, well controlled, sincerely declaring the already known limitations that will have a significant impact on molecular and developmental studies in cells and in animals.
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Reviewer #1 (Public Review):
In this paper, Macdonald et al describe an original method for the inactivation of gene function using auxin-inducible degrons. They validate their approach in mice in this proof of concept on two genes: Ncaph and Ncaph2. The paper shows the efficiency of knockdown at the protein level both in mice and in different primary cells. It also shows the rapidity of degradation of the targeted protein.
An immediate parallel comes between this technology and the cre and creERT2 spatiotemporal approach which has revolutionized the study of gene function in a cell/tissue type, in adults (bypassing embryonic lethality) and also allows to dissect more finely the different functions of pleiotropic genes. By acting at the protein level, this auxin-inducible degrons approach opens new solutions complementary to the …
Reviewer #1 (Public Review):
In this paper, Macdonald et al describe an original method for the inactivation of gene function using auxin-inducible degrons. They validate their approach in mice in this proof of concept on two genes: Ncaph and Ncaph2. The paper shows the efficiency of knockdown at the protein level both in mice and in different primary cells. It also shows the rapidity of degradation of the targeted protein.
An immediate parallel comes between this technology and the cre and creERT2 spatiotemporal approach which has revolutionized the study of gene function in a cell/tissue type, in adults (bypassing embryonic lethality) and also allows to dissect more finely the different functions of pleiotropic genes. By acting at the protein level, this auxin-inducible degrons approach opens new solutions complementary to the cre/loxP system to achieve these three objectives.
As with any new tool, there are at least two main questions to answer regarding the use of auxin-inducible degrons:
• Is this technology effective, in this case, does it efficiently reduce target protein expression?
• What are the biases of this new technology?Concerning the use of the cre/loxP system, many years and many articles have been necessary to better master the technology and especially to better understand its biases and how to bypass them. It is therefore unrealistic to expect this article to completely answer the two questions above. The term proof of concept seems thus interesting to put forward in this article. Indeed, this paper is above all a proof of the effectiveness of this system on two genes. Regarding the description of the biases, it provides elements that suggest that some drawbacks, similar to those of the creERT2 system, could be expected. In the future, additional papers will be required to better characterize the tool and understand for which target gene this approach is efficient and specific.
Strengths:
• This paper is a proof of concept of the efficiency of this approach for inactivation at the protein level.
• It induces a rapid target protein degradation which allows the immediate study of a phenotype.
• The methods of validation of the auxin-inducible degrons approach are convincing.
• The results are very complementary to the paper of Yesbolatova et al. and thus open technological opportunities for the use of this system in cell lines and animal models, particularly the mouse.
• The whole text is very precise and well detailed (including materials and methods and supplementary figures) facilitating reuse by other scientists.
• The proteome data are available in the PRIDE repository, highlighting the authors' commitment to FAIR principles.Weaknesses:
• Potential biases and how to work around them should be discussed further in this paper and will require additional studies in the future.
• The level of generalizability of this approach will require further studies.
• This approach only addresses the study of protein-coding genes and will not work to study the non-coding genome. -
