Curing “GFP-itis” in Bacteria with Base Editors: Development of a Genome Editing Science Program Implemented with High School Biology Students
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (Arcadia Science)
Abstract
The flexibility and precision of CRISPR-Cas9 and related technologies have made these genome editing tools increasingly popular in agriculture, medicine, and basic science research over the past decade. Genome editing will continue to be relevant and utilized across diverse scientific fields in the future. Given this, students should be introduced to genome editing technologies and encouraged to consider their ethical implications early on in pre-college biology curricula. Furthermore, instruction on this topic presents an opportunity to create partnerships between researchers and educators at the K-12 levels that can strengthen student engagement in science, technology, engineering, and mathematics (STEM). To this end, we present a three-day student-centered learning program to introduce high school students to genome editing technologies through a hands-on base editing experiment in E. coli , accompanied by a relevant background lecture and facilitated ethics discussion. This unique partnership aims to educate students and provides a framework for research institutions to implement genome editing outreach programs at local high schools.
Article activity feed
-
provides a framework for research institutions to implement genome editing outreach programs at local high schools
In "Conclusions and Future Outlook," you note that you created this program and are presumably sharing this paper to help other genome editing researchers implement partnerships with local schools. This is awesome and I think you could make it clearer in the abstract. Since this is most readers' first introduction to your content, it's a good place to specify your intended audience and emphasize that you've included all the resources necessary for them to replicate or adapt your program.
-
A. Current base editor technologies have the capacity to correct a large fraction of human pathogenic single nucleotide variants back to wild-type.
I think this could be clearer in specifying what exactly you're depicting in panel A
-
Current base editors use a common architecture to achieve single base conversions. Cas9n (blue) in combination with a targeting gRNA (green) directs the editor to the genomic site of interest by base-pairing with the protospacer (orange) sequence. A protospacer adjacent motif (PAM, blue) is also required for Cas9:DNA binding. This then allows the ssDNA modifying enzyme (red) to chemically modify a target base of interest within a small window of exposed ssDNA (yellow). Overall base pair conversions (listed in the table at the bottom) are determined by the nature of the ssDNA enzyme, and the inclusion of DNA repair manipulation components (purple).
Perhaps cite a review that general genome editing researchers might read to get more familiar with base editing should they choose to implement your program.
-
Figure 2.
This could be a little cleaner (italicize E. coli, add degree symbol, etc.). At a glance, I'm not sure what the italicized purple letters in the pSel panel are supposed to indicate.
-
Genome engineering (or genome editing) is the manipulation of the genomic sequence of a living organism through the addition, deletion, correction, or replacement of DNA in a precise, efficient, and controllable manner.
This definition is a little misleading. Perhaps rephrase: "Genome engineering (or genome editing) is the manipulation of the genomic sequence of a living organism through the addition, deletion, correction, or replacement of DNA. Ideally, this occurs in a precise, efficient, and controllable manner."
-
germline genome editing
I know germline editing is the go-to ethics discussion topic, but I wonder if you might engage students more deeply by discussing the concept of enhancement. This could tie in really well with the hands-on experiment because adding GFP to E. coli is itself an enhancement.
You can make allusions to superheroes, which tends to hook younger audiences. Imagine adding GFP to a person and knocking out myostatin... could we make the Incredible Hulk?
You could also discuss whether enhanced people should be able to compete in professional sports, how genetic enhancement does/doesn't differ from plastic surgery or tattoos, etc. Lots of ground for connection with high school students.
-
Kudos for developing this program, I know how hard it is to make these topics accessible to lay audiences! This manuscript does a great job of articulating the need for practicing scientists to engage with students and provides a useful framework for others to do so.
My main feedback is that you could make the content even more useful by including practical details and tips. For example, how do you recommend that scientists get in touch with local educators? Do you recommend a small trial run of just the experiment before implementing the full program with multiple classes? What is an effective ratio of scientist volunteers to students? In the materials list, what quantity of each material is required per student group? What were your main challenges as instructors and how do you recommend that others overcome them?
I think anything …
Kudos for developing this program, I know how hard it is to make these topics accessible to lay audiences! This manuscript does a great job of articulating the need for practicing scientists to engage with students and provides a useful framework for others to do so.
My main feedback is that you could make the content even more useful by including practical details and tips. For example, how do you recommend that scientists get in touch with local educators? Do you recommend a small trial run of just the experiment before implementing the full program with multiple classes? What is an effective ratio of scientist volunteers to students? In the materials list, what quantity of each material is required per student group? What were your main challenges as instructors and how do you recommend that others overcome them?
I think anything that can make this process less intimidating will encourage readers to commit to such a time-consuming but rewarding effort. I've also made annotated comments throughout that pertain to this (and other topics).
I also wonder if you might make the slides and worksheet available in editable format so that they're easy for readers to use and change. It would be great to include a link to a publicly viewable/downloadable Google Slides version of the deck and Google Doc version of the worksheet (and possibly other supplemental resources).
-
are typically
Suggest replacing with "may be" since there are others in the field who might disagree
-
We have since improved the worksheet using the students’ feedback by further clarifying some questions and updating instructions for labelling diagrams
I think the diagram could be a little clearer and maybe it would help the students understand better. For example, making the DNA and RNA strands different colors would make them easier to quickly distinguish. Same for the Cas9 protein vs. the DNA-modifying enzyme. You could also put a box around the target base location just to make it very clear where that empty label box is pointing.
I would make sure that however you depict the system in the worksheet, it's identical to how it's shown in the slides.
-
GFP-itis
This is not a hugely important comment, but "GFP-itis" feels awkward to pronounce out loud, and also feels a little inaccurate because GFP is not something E. coli would normally express. I wonder if you might brainstorm some other names, like "Chronic blandness" or "Swag deficiency" or something that's still light-hearted but easy to pronounce.
-
this
Unclear what "this" refers to, perhaps replace with "the dead GFP"
-
The lowest rating was observed for the lecture, which overall scored a 3.6 out of 5 (n=59), with 50 students indicating a 3 or higher. In the open comments section, several students commented favorably on the active learning elements of the lecture, prompting us to consider including more of these strategies in future designs.
I would definitely encourage more active learning! Or perhaps replacing some of the slides about base editing with an animation could make them a bit clearer. It's definitely a lot for students to wrap their heads around basic genome editing, let alone base editing. I wonder if you could get away with skipping traditional editing just to reduce the complexity.
-
addgene: 195342
Very glad to see these plasmids already available on Addgene! That's crucial to make this replicable
-
by treating them with an ABE
Would add "and restoring fluorescence."
-
Supplemental Information
Would be nice to have the supplemental info as part of the preprint, not sure if you can just combine the PDFs and upload
-
Notably, we include here requisite materials for others to reproduce our program with their local communities.
👏
-
full protocol
I think you could write the instructions in a simpler way for the students, especially since you note that administering this portion of the program is the most challenging. Breaking the protocol up into short, imperative steps might help.
For example, Step 1 can just be labeling the tubes and putting them on ice. Step 2 can be "Add 70 ul sterile DI water and 20 ul 5x KCM to each tube." etc.
-
We list recommended portable equipment in the SI if the classroom does not have access to certain equipment.
Is there any way this might work without a water bath or incubator? These seem the most unlikely for a HS to have and toughest to bring with you from lab. Wondering if maybe you can leave plates out at room temp for a weekend to grow instead. Not essential, just a thought
-
However, we did not assess this knowledge in a pre-post format.
This would be great to see. I'm extremely dubious that so many students are actually well-informed or even aware of genome editing/CRISPR/base editing.
-
research-practice partnership such as this strengthen ties between academia and their communities and provide opportunities for students to nourish their interests in STEM
It would be really cool to poll your scientist instructors and learn how they benefitted from the experience too. Even an informal discussion could be interesting to include, especially if you're hoping to encourage readers to do this themselves.
-
To adapt this program for a more advanced class, such as an undergraduate laboratory course unit, instructors could emphasize the role of designing genome editing tools and ask students to generate their own gRNAs to correct the dGFP sequence
A lot of advanced classrooms/educators may want to verify the edit at the genetic level (this optional extension is available in many of the traditional DSB-mediated editing courses I've seen). This is probably excessive, but perhaps you might suggest primers to amplify a region that includes the editing site and suggest a restriction enzyme that will only cut (or only not cut) if editing is successful.
-
Figure 2.
This could be a little cleaner (italicize E. coli, add degree symbol, etc.). At a glance, I'm not sure what the italicized purple letters in the pSel panel are supposed to indicate.
-
Current base editors use a common architecture to achieve single base conversions. Cas9n (blue) in combination with a targeting gRNA (green) directs the editor to the genomic site of interest by base-pairing with the protospacer (orange) sequence. A protospacer adjacent motif (PAM, blue) is also required for Cas9:DNA binding. This then allows the ssDNA modifying enzyme (red) to chemically modify a target base of interest within a small window of exposed ssDNA (yellow). Overall base pair conversions (listed in the table at the bottom) are determined by the nature of the ssDNA enzyme, and the inclusion of DNA repair manipulation components (purple).
Perhaps cite a review that general genome editing researchers might read to get more familiar with base editing should they choose to implement your program.
-
A. Current base editor technologies have the capacity to correct a large fraction of human pathogenic single nucleotide variants back to wild-type.
I think this could be clearer in specifying what exactly you're depicting in panel A
-
To adapt this program for a more advanced class, such as an undergraduate laboratory course unit, instructors could emphasize the role of designing genome editing tools and ask students to generate their own gRNAs to correct the dGFP sequence
A lot of advanced classrooms/educators may want to verify the edit at the genetic level (this optional extension is available in many of the traditional DSB-mediated editing courses I've seen). This is probably excessive, but perhaps you might suggest primers to amplify a region that includes the editing site and suggest a restriction enzyme that will only cut (or only not cut) if editing is successful.
-
However, we did not assess this knowledge in a pre-post format.
This would be great to see. I'm extremely dubious that so many students are actually well-informed or even aware of genome editing/CRISPR/base editing.
-
Notably, we include here requisite materials for others to reproduce our program with their local communities.
👏
-
research-practice partnership such as this strengthen ties between academia and their communities and provide opportunities for students to nourish their interests in STEM
It would be really cool to poll your scientist instructors and learn how they benefitted from the experience too. Even an informal discussion could be interesting to include, especially if you're hoping to encourage readers to do this themselves.
-
germline genome editing
I know germline editing is the go-to ethics discussion topic, but I wonder if you might engage students more deeply by discussing the concept of enhancement. This could tie in really well with the hands-on experiment because adding GFP to E. coli is itself an enhancement.
You can make allusions to superheroes, which tends to hook younger audiences. Imagine adding GFP to a person and knocking out myostatin... could we make the Incredible Hulk?
You could also discuss whether enhanced people should be able to compete in professional sports, how genetic enhancement does/doesn't differ from plastic surgery or tattoos, etc. Lots of ground for connection with high school students.
-
We have since improved the worksheet using the students’ feedback by further clarifying some questions and updating instructions for labelling diagrams
I think the diagram could be a little clearer and maybe it would help the students understand better. For example, making the DNA and RNA strands different colors would make them easier to quickly distinguish. Same for the Cas9 protein vs. the DNA-modifying enzyme. You could also put a box around the target base location just to make it very clear where that empty label box is pointing.
I would make sure that however you depict the system in the worksheet, it's identical to how it's shown in the slides.
-
The lowest rating was observed for the lecture, which overall scored a 3.6 out of 5 (n=59), with 50 students indicating a 3 or higher. In the open comments section, several students commented favorably on the active learning elements of the lecture, prompting us to consider including more of these strategies in future designs.
I would definitely encourage more active learning! Or perhaps replacing some of the slides about base editing with an animation could make them a bit clearer. It's definitely a lot for students to wrap their heads around basic genome editing, let alone base editing. I wonder if you could get away with skipping traditional editing just to reduce the complexity.
-
We list recommended portable equipment in the SI if the classroom does not have access to certain equipment.
Is there any way this might work without a water bath or incubator? These seem the most unlikely for a HS to have and toughest to bring with you from lab. Wondering if maybe you can leave plates out at room temp for a weekend to grow instead. Not essential, just a thought
-
full protocol
I think you could write the instructions in a simpler way for the students, especially since you note that administering this portion of the program is the most challenging. Breaking the protocol up into short, imperative steps might help.
For example, Step 1 can just be labeling the tubes and putting them on ice. Step 2 can be "Add 70 ul sterile DI water and 20 ul 5x KCM to each tube." etc.
-
Supplemental Information
Would be nice to have the supplemental info as part of the preprint, not sure if you can just combine the PDFs and upload
-
addgene: 195342
Very glad to see these plasmids already available on Addgene! That's crucial to make this replicable
-
by treating them with an ABE
Would add "and restoring fluorescence."
-
GFP-itis
This is not a hugely important comment, but "GFP-itis" feels awkward to pronounce out loud, and also feels a little inaccurate because GFP is not something E. coli would normally express. I wonder if you might brainstorm some other names, like "Chronic blandness" or "Swag deficiency" or something that's still light-hearted but easy to pronounce.
-
this
Unclear what "this" refers to, perhaps replace with "the dead GFP"
-
are typically
Suggest replacing with "may be" since there are others in the field who might disagree
-
Genome engineering (or genome editing) is the manipulation of the genomic sequence of a living organism through the addition, deletion, correction, or replacement of DNA in a precise, efficient, and controllable manner.
This definition is a little misleading. Perhaps rephrase: "Genome engineering (or genome editing) is the manipulation of the genomic sequence of a living organism through the addition, deletion, correction, or replacement of DNA. Ideally, this occurs in a precise, efficient, and controllable manner."
-
Kudos for developing this program, I know how hard it is to make these topics accessible to lay audiences! This manuscript does a great job of articulating the need for practicing scientists to engage with students and provides a useful framework for others to do so.
My main feedback is that you could make the content even more useful by including practical details and tips. For example, how do you recommend that scientists get in touch with local educators? Do you recommend a small trial run of just the experiment before implementing the full program with multiple classes? What is an effective ratio of scientist volunteers to students? In the materials list, what quantity of each material is required per student group? What were your main challenges as instructors and how do you recommend that others overcome them?
I think anything …
Kudos for developing this program, I know how hard it is to make these topics accessible to lay audiences! This manuscript does a great job of articulating the need for practicing scientists to engage with students and provides a useful framework for others to do so.
My main feedback is that you could make the content even more useful by including practical details and tips. For example, how do you recommend that scientists get in touch with local educators? Do you recommend a small trial run of just the experiment before implementing the full program with multiple classes? What is an effective ratio of scientist volunteers to students? In the materials list, what quantity of each material is required per student group? What were your main challenges as instructors and how do you recommend that others overcome them?
I think anything that can make this process less intimidating will encourage readers to commit to such a time-consuming but rewarding effort. I've also made annotated comments throughout that pertain to this (and other topics).
I also wonder if you might make the slides and worksheet available in editable format so that they're easy for readers to use and change. It would be great to include a link to a publicly viewable/downloadable Google Slides version of the deck and Google Doc version of the worksheet (and possibly other supplemental resources).
-
provides a framework for research institutions to implement genome editing outreach programs at local high schools
In "Conclusions and Future Outlook," you note that you created this program and are presumably sharing this paper to help other genome editing researchers implement partnerships with local schools. This is awesome and I think you could make it clearer in the abstract. Since this is most readers' first introduction to your content, it's a good place to specify your intended audience and emphasize that you've included all the resources necessary for them to replicate or adapt your program.
-
