On demand expression control of endogenous genes with DExCon, DExogron and LUXon reveals differential dynamics of Rab11 family members
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Evaluation Summary:
This work reports a genome editing strategy that enables blocking and tetracycline-controlled re-expression of fluorescently-tagged proteins from endogenous loci. The authors combine this with the photoactivatable tet-on/off system, a knocksideways approach, as well as the auxin-inducible degron system to improve spatial and temporal control of gene expression. They demonstrate the applicability of the methods by studying the localization, function and protein-expression dynamics of the Rab11-family of small GTPases. Using these approaches, the authors discover subtle differences in the localization as well as expression and degradation kinetics of Rab11a/b/Rab25. The work will be of broad interest to molecular and cell biologists.
(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 #2 agreed to share their name with the authors.)
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Abstract
CRISPR technology has made generation of gene knock-outs widely achievable in cells. However, once inactivated, their re-activation remains difficult, especially in diploid cells. Here, we present DExCon ( D oxycycline-mediated endogenous gene Ex pression Con trol), DExogron (DExCon combined with auxin-mediated targeted protein degradation), and LUXon (light responsive DExCon) approaches which combine one-step CRISPR-Cas9-mediated targeted knockin of fluorescent proteins with an advanced Tet-inducible TRE3GS promoter. These approaches combine blockade of active gene expression with the ability to re-activate expression on demand, including activation of silenced genes. Systematic control can be exerted using doxycycline or spatiotemporally by light, and we demonstrate functional knock-out/rescue in the closely related Rab11 family of vesicle trafficking regulators. Fluorescent protein knock-in results in bright signals compatible with low-light live microscopy from monoallelic modification, the potential to simultaneously image different alleles of the same gene, and bypasses the need to work with clones. Protein levels are easily tunable to correspond with endogenous expression through cell sorting (DExCon), timing of light illumination (LUXon), or by exposing cells to different levels of auxin (DExogron). Furthermore, our approach allowed us to quantify previously unforeseen differences in vesicle dynamics, transferrin receptor recycling, expression kinetics, and protein stability among highly similar endogenous Rab11 family members and their colocalization in triple knock-in ovarian cancer cell lines.
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Author Response
Reviewer #1 (Public Review):
The genome-editing strategies presented here represent a fantastic technology pipeline, comprehensively tested and precious to the cell biology field. While I am positive about the value of this contribution, I have three major requests that require some experimental work to make the study truly convincing and comprehensible.
- The DExCon system allows re-expression of N-terminal tagged proteins from the endogenous locus and, in theory, should allow re-expression of all protein-coding splicing isoforms. This provides an advantage over generation of a KO cell line and subsequent tet-inducible rescue from a viral vector containing cDNA. This is undeniably an important technical advantage because it can potentially recapitulate the spectrum of functions of the locus. However, the authors do …
Author Response
Reviewer #1 (Public Review):
The genome-editing strategies presented here represent a fantastic technology pipeline, comprehensively tested and precious to the cell biology field. While I am positive about the value of this contribution, I have three major requests that require some experimental work to make the study truly convincing and comprehensible.
- The DExCon system allows re-expression of N-terminal tagged proteins from the endogenous locus and, in theory, should allow re-expression of all protein-coding splicing isoforms. This provides an advantage over generation of a KO cell line and subsequent tet-inducible rescue from a viral vector containing cDNA. This is undeniably an important technical advantage because it can potentially recapitulate the spectrum of functions of the locus. However, the authors do not provide direct evidence that the DExCon system does allow for re-expression of multiple splicing isoforms. One suggestion would be to identify the Rab11 splice variants expressed in A2780 cells and demonstrate that the relative abundance of these splice variants is not altered upon fluorescent-tagging and CMV-promotor-driven overexpression of Rab11 from the endogenous locus. This seems to me to be a crucial result to demonstrate the effectiveness of the method.
We thank for this great suggestion and have included new data to answer this as described in Figure 2 figure supplement 1 and the text on page 5 of the new manuscript.
- The authors use a CMV-promotor to rescue of Rab11/25 gene expression. They convincingly show that it is possible to tune expression levels by FACS sorting. However, for most experiments, the authors use expression levels of Rab11a/b/Rab25 that are much higher than endogenous levels. Since high expression levels of Rab11a/b can affect its localization (transient expression Fig 2G), they should show that the Rab11a/b/Rab25 expression levels used do not alter localization and function. This could be tested simply by a transferrin recycling assay. To ensure that DExCon-Rab11/25 expression levels do not affect localization, the authors could use cells containing a knock-in of mCH-Rab11a on one allele and DExCon-mNG-Rab11a on the other allele and compare their localization.
We thank the reviewer for these important and interesting suggestions, which have been answered in a new Figure 7 and new Figure 5 figure supplement 2.
- In Fig 6F, the effect of Rab11 on migration is tested using DExogron-mCH-R11b in a wound healing assay. Loss of R11b expression by DExogron-mCH-R11b reduced migration and this effect could be rescued by dox-induced expression of DExogron-mCH-R11b. However, IAA treatment failed to prevent this rescue as would have been expected. The authors hypothesize that this results from incomplete protein degradation under +dox +IAA conditions. In Fig 6K the authors solve this problem by removing dox when treating with IAA. The authors should repeat the experiment 6F under -dox +IAA conditions.
This is an important point and we have addressed this in a new Figure 6 figure supplement 2F and in the text on page 17.
Reviewer #2 (Public Review):
This is a very interesting, quite dense study that reports several new techniques of controlling cellular protein levels, as well as performing spatiotemporal image analysis. The strength of this study is the combination of various previously known approaches (like CRISPR knock-in, Degron, knock-sideways) to allow quite precise control of protein levels (by controlling degradation or expression), as well as imaging of endogenously tagged proteins. The ability to inactivate/reactivate proteins of interest is a huge achievement that will be very useful in many studies by this and other laboratories. Another big strength of this study is the fact that the authors took time to optimize and streamline these approaches making them much more user-friendly as compared to earlier versions of many of these approaches. The only very minor drawback of this manuscript is the fact that authors have chosen to perform proof-of-principal studies using Rab11 family of proteins (which is great) but in rather boring cell types. Rab11 family members are presumably involved in differentially regulating various aspects of cell polarity and recycling. Thus, it's not too surprising that authors did not see that many differences in rab11a, rab11b and rab25 functions since they used a single cargo (transferrin) and non-polarized cancer cells. However, I do realize that the main goal of this study is not to investigate Rab11 but rather to develop new techniques. Thus, this minor weakness should not stop this manuscript from being published.
We thank the Reviewer 2 for support and suggestions. A2780 ovarian cancer cells were selected as an established model of migration and invasion. They polarize in 3D matrix, so we can ask questions about the functions of Rab11 family members in these processes, and explore the full range of gene expression control (dox/degron/light). We anticipate our methods will be tractable to other cell types and look forward to ourselves and others using these in different biological contexts.
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Evaluation Summary:
This work reports a genome editing strategy that enables blocking and tetracycline-controlled re-expression of fluorescently-tagged proteins from endogenous loci. The authors combine this with the photoactivatable tet-on/off system, a knocksideways approach, as well as the auxin-inducible degron system to improve spatial and temporal control of gene expression. They demonstrate the applicability of the methods by studying the localization, function and protein-expression dynamics of the Rab11-family of small GTPases. Using these approaches, the authors discover subtle differences in the localization as well as expression and degradation kinetics of Rab11a/b/Rab25. The work will be of broad interest to molecular and cell biologists.
(This preprint has been reviewed by eLife. We include the public reviews from the …
Evaluation Summary:
This work reports a genome editing strategy that enables blocking and tetracycline-controlled re-expression of fluorescently-tagged proteins from endogenous loci. The authors combine this with the photoactivatable tet-on/off system, a knocksideways approach, as well as the auxin-inducible degron system to improve spatial and temporal control of gene expression. They demonstrate the applicability of the methods by studying the localization, function and protein-expression dynamics of the Rab11-family of small GTPases. Using these approaches, the authors discover subtle differences in the localization as well as expression and degradation kinetics of Rab11a/b/Rab25. The work will be of broad interest to molecular and cell biologists.
(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 #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The genome-editing strategies presented here represent a fantastic technology pipeline, comprehensively tested and precious to the cell biology field. While I am positive about the value of this contribution, I have three major requests that require some experimental work to make the study truly convincing and comprehensible.
1. The DExCon system allows re-expression of N-terminal tagged proteins from the endogenous locus and, in theory, should allow re-expression of all protein-coding splicing isoforms. This provides an advantage over generation of a KO cell line and subsequent tet-inducible rescue from a viral vector containing cDNA. This is undeniably an important technical advantage because it can potentially recapitulate the spectrum of functions of the locus. However, the authors do not provide direct …
Reviewer #1 (Public Review):
The genome-editing strategies presented here represent a fantastic technology pipeline, comprehensively tested and precious to the cell biology field. While I am positive about the value of this contribution, I have three major requests that require some experimental work to make the study truly convincing and comprehensible.
1. The DExCon system allows re-expression of N-terminal tagged proteins from the endogenous locus and, in theory, should allow re-expression of all protein-coding splicing isoforms. This provides an advantage over generation of a KO cell line and subsequent tet-inducible rescue from a viral vector containing cDNA. This is undeniably an important technical advantage because it can potentially recapitulate the spectrum of functions of the locus. However, the authors do not provide direct evidence that the DExCon system does allow for re-expression of multiple splicing isoforms. One suggestion would be to identify the Rab11 splice variants expressed in A2780 cells and demonstrate that the relative abundance of these splice variants is not altered upon fluorescent-tagging and CMV-promotor-driven overexpression of Rab11 from the endogenous locus. This seems to me to be a crucial result to demonstrate the effectiveness of the method.
2. The authors use a CMV-promotor to rescue of Rab11/25 gene expression. They convincingly show that it is possible to tune expression levels by FACS sorting. However, for most experiments, the authors use expression levels of Rab11a/b/Rab25 that are much higher than endogenous levels. Since high expression levels of Rab11a/b can affect its localization (transient expression Fig 2G), they should show that the Rab11a/b/Rab25 expression levels used do not alter localization and function. This could be tested simply by a transferrin recycling assay. To ensure that DExCon-Rab11/25 expression levels do not affect localization, the authors could use cells containing a knock-in of mCH-Rab11a on one allele and DExCon-mNG-Rab11a on the other allele and compare their localization.
3. In Fig 6F, the effect of Rab11 on migration is tested using DExogron-mCH-R11b in a wound healing assay. Loss of R11b expression by DExogron-mCH-R11b reduced migration and this effect could be rescued by dox-induced expression of DExogron-mCH-R11b. However, IAA treatment failed to prevent this rescue as would have been expected. The authors hypothesize that this results from incomplete protein degradation under +dox +IAA conditions. In Fig 6K the authors solve this problem by removing dox when treating with IAA. The authors should repeat the experiment 6F under -dox +IAA conditions.
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Reviewer #2 (Public Review):
This is a very interesting, quite dense study that reports several new techniques of controlling cellular protein levels, as well as performing spatiotemporal image analysis. The strength of this study is the combination of various previously known approaches (like CRISPR knock-in, Degron, knock-sideways) to allow quite precise control of protein levels (by controlling degradation or expression), as well as imaging of endogenously tagged proteins. The ability to inactivate/reactivate proteins of interest is a huge achievement that will be very useful in many studies by this and other laboratories. Another big strength of this study is the fact that the authors took time to optimize and streamline these approaches making them much more user-friendly as compared to earlier versions of many of these approaches. …
Reviewer #2 (Public Review):
This is a very interesting, quite dense study that reports several new techniques of controlling cellular protein levels, as well as performing spatiotemporal image analysis. The strength of this study is the combination of various previously known approaches (like CRISPR knock-in, Degron, knock-sideways) to allow quite precise control of protein levels (by controlling degradation or expression), as well as imaging of endogenously tagged proteins. The ability to inactivate/reactivate proteins of interest is a huge achievement that will be very useful in many studies by this and other laboratories. Another big strength of this study is the fact that the authors took time to optimize and streamline these approaches making them much more user-friendly as compared to earlier versions of many of these approaches. The only very minor drawback of this manuscript is the fact that authors have chosen to perform proof-of-principal studies using Rab11 family of proteins (which is great) but in rather boring cell types. Rab11 family members are presumably involved in differentially regulating various aspects of cell polarity and recycling. Thus, it's not too surprising that authors did not see that many differences in rab11a, rab11b and rab25 functions since they used a single cargo (transferrin) and non-polarized cancer cells. However, I do realize that the main goal of this study is not to investigate Rab11 but rather to develop new techniques.
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