Endogenous GFP tagging in the diatom Thalassiosira pseudonana
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Abstract
The regulated abundance and spatial distribution of proteins determines cellular structure and function. The discovery of green fluorescent protein (GFP) and fusing it to a target protein to determine subcellular localization revolutionized cell biology. Most localization studies involve introducing additional copies of a target gene genetically fused to GFP and under the control of a constitutive promoter, resulting in the expression of the GFP-fusion protein at non-native levels. Here we have developed a single vector CRISPR/Cas9 guided GFP knock-in strategy in the diatom Thalassiosira pseudonana . This enables precise and scarless knock-in of GFP at the endogenous genomic location to create GFP fusion proteins under their native cis and trans regulatory elements with knock-in efficiencies of over 50%. We show that a previously uncharacterized bestrophin-like protein localizes to the CO 2 -fixing pyrenoid and demonstrate that by measuring GFP fluorescence we can track relative protein abundance in response to environmental change. To enable endogenous tagging, we developed a Golden Gate Molecular Cloning system for the rapid assembly of episomes for transformation into Thalassiosira pseudonana via bacterial conjugation. In addition, this versatile toolbox enables CRISPR/Cas9 gene editing, provides a broad range of validated fluorophores and enables future large-scale functional studies in diatoms.
Significance statement
Fluorescent protein (FP) tagging is a widely utilized technique for understanding the spatial distribution of proteins. However, introducing extra gene copies under constitutive promoters that randomly integrate into the genome can result in non-biologically relevant expression levels, unwanted genomic mutations and localization artefacts. To overcome this, we developed a novel single vector system capable of CRISPR/Cas9-guided endogenous GFP tagging in a globally important model diatom. This allows scarless GFP knock-in at precise genomic locations resulting in GFP fusions regulated by native promoters/terminators, which facilitates accurate localization and determination of relative protein abundance. Moreover, the developed modular cloning framework is user-friendly and opens the door for high throughput large-scale studies, including FP tagging, knock-out, and knock-in.
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In addition, exploring the limitations of knock-in fragment size would be useful to determine if the approach could be used for multi-gene/pathway knock-in at neutral genomic sites for biotechnology applications.
one idea - you could explore split FPs - you could express GFP(1-10) off the episome and then just KI in GFP11's (there are several split FPs now available) - this could be a rapid way to label multiple things (though you would need to maintain the line using NAT selection) - which brings me to my last question - have you thought about making a landing pad for site specific insertion now that you can insert into defined locations? There would be all sorts of great use cases for this!
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It has been demonstrated in other organisms that HDR can be offset from the cut-site which would then enable GFP to be inserted directly prior to the stop codon and increase sgRNA flexibility. However, knock-in efficiency can rapidly drop off as the cut-site to HDR-site distance increases39.
Yes! See my comment above - I definitely think, given the kind of efficiency you report here, that you can explore moving your insertion site to the N- or C-terminus of your target gene
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Using CRISPick28 (see methods) we selected a sgRNA with a predicted on-target score of 0.7 that cut 40 bp upstream of the BST2 stop codon. We designed homology arms that covered both sides of the predicted Cas9 cut site (Fig. 2a)
Based on lots of tagging of endogenous loci using a similar plasmid based delivery system in C. elegans, I would think that you should be able to get away with moving the insertion site to the C-terminus (in this case) or N-terminus (in other cases) - it would be great to test this out - but we (and others) have found that if you re-code the homology arm in your repair plasmid between the cut site and insertion point, you can get KIs when you push the distance between the cut site and the insertion site, avoiding having to insert your FP into the gene. Is there a locus of interest with some different sgRNAs …
Using CRISPick28 (see methods) we selected a sgRNA with a predicted on-target score of 0.7 that cut 40 bp upstream of the BST2 stop codon. We designed homology arms that covered both sides of the predicted Cas9 cut site (Fig. 2a)
Based on lots of tagging of endogenous loci using a similar plasmid based delivery system in C. elegans, I would think that you should be able to get away with moving the insertion site to the C-terminus (in this case) or N-terminus (in other cases) - it would be great to test this out - but we (and others) have found that if you re-code the homology arm in your repair plasmid between the cut site and insertion point, you can get KIs when you push the distance between the cut site and the insertion site, avoiding having to insert your FP into the gene. Is there a locus of interest with some different sgRNAs at the C- or N-terminus that you could experimentally try this out with (like one that would split the insertion site, one that would be ~10-20 bp away, and one that would be ~50-60bp away)?
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It has been demonstrated in other organisms that HDR can be offset from the cut-site which would then enable GFP to be inserted directly prior to the stop codon and increase sgRNA flexibility. However, knock-in efficiency can rapidly drop off as the cut-site to HDR-site distance increases39.
Yes! See my comment above - I definitely think, given the kind of efficiency you report here, that you can explore moving your insertion site to the N- or C-terminus of your target gene
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In addition, exploring the limitations of knock-in fragment size would be useful to determine if the approach could be used for multi-gene/pathway knock-in at neutral genomic sites for biotechnology applications.
one idea - you could explore split FPs - you could express GFP(1-10) off the episome and then just KI in GFP11's (there are several split FPs now available) - this could be a rapid way to label multiple things (though you would need to maintain the line using NAT selection) - which brings me to my last question - have you thought about making a landing pad for site specific insertion now that you can insert into defined locations? There would be all sorts of great use cases for this!
-
Using CRISPick28 (see methods) we selected a sgRNA with a predicted on-target score of 0.7 that cut 40 bp upstream of the BST2 stop codon. We designed homology arms that covered both sides of the predicted Cas9 cut site (Fig. 2a)
Based on lots of tagging of endogenous loci using a similar plasmid based delivery system in C. elegans, I would think that you should be able to get away with moving the insertion site to the C-terminus (in this case) or N-terminus (in other cases) - it would be great to test this out - but we (and others) have found that if you re-code the homology arm in your repair plasmid between the cut site and insertion point, you can get KIs when you push the distance between the cut site and the insertion site, avoiding having to insert your FP into the gene. Is there a locus of interest with some different sgRNAs …
Using CRISPick28 (see methods) we selected a sgRNA with a predicted on-target score of 0.7 that cut 40 bp upstream of the BST2 stop codon. We designed homology arms that covered both sides of the predicted Cas9 cut site (Fig. 2a)
Based on lots of tagging of endogenous loci using a similar plasmid based delivery system in C. elegans, I would think that you should be able to get away with moving the insertion site to the C-terminus (in this case) or N-terminus (in other cases) - it would be great to test this out - but we (and others) have found that if you re-code the homology arm in your repair plasmid between the cut site and insertion point, you can get KIs when you push the distance between the cut site and the insertion site, avoiding having to insert your FP into the gene. Is there a locus of interest with some different sgRNAs at the C- or N-terminus that you could experimentally try this out with (like one that would split the insertion site, one that would be ~10-20 bp away, and one that would be ~50-60bp away)?
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