Endogenous tagging using split mNeonGreen in human iPSCs for live imaging studies

Reviewer #1 (Public Review):

Summary:
In this manuscript, the authors have applied an asymmetric split mNeonGreen2 (mNG2) system to human iPSCs. Integrating a constitutively expressed long fragment of mNG2 at the AAVS1 locus, allows other proteins to be tagged through the use of available ssODN donors. This removes the need to generate long AAV donors for tagging, thus greatly facilitating high-throughput tagging efforts. The authors then demonstrate the feasibility of the method by successfully tagging 9 markers expressed in iPSC at various, and one expressed upon endoderm differentiation. Several additional differentiation markers were also successfully tagged but not subsequently tested for expression/visibility. As one might expect for high-throughput tagging, a few proteins, while successfully tagged at the genomic level, failed to be visible. Finally, to demonstrate the utility of the tagged cells, the authors isolated clones with genes relevant to cytokinesis tagged, and together with an AI to enhance signal-to-noise ratios, monitored their localization over cell division.

Strengths:
Characterization of the mNG2 tagged parental iPSC line was well and carefully done including validation of a single integration, the presence of markers for continued pluripotency, selected off-target analysis, and G-banding-based structural rearrangement detection.

The ability to tag proteins with simple ssODNs in iPSC capable of multi-lineage differentiation will undoubtedly be useful for localization tracking and reporter line generation.

Validation of clone genotypes was carefully performed and highlights the continued need for caution with regard to editing outcomes.

Weaknesses:
IF and flow cytometry figures lack quantification and information on replication. How consistent is the brightness and localization of the markers? How representative are the specific images? Stability is mentioned in the text but data on the stability of expression/brightness is not shown.

The localization of markers, while consistent with expectations, is not validated by a second technique such as antibody staining, and in many cases not even with Hoechst to show nuclear vs cytoplasmic.

For the multi-germ layer differentiation validation, NCAM is also expressed by ectoderm, so isn't a good solo marker for mesoderm as it was used. Indeed, the kit used for the differentiation suggests Brachyury combined with either NCAM or CXCR4, not NCAM alone.

Only a single female parental line has been generated and characterized. It would have been useful to have several lines and both male and female to allow sex differences to be explored.

The AI-based signal-to-noise enhancement needs more details and testing. Such models can introduce strong assumptions and thus artefacts into the resolved data. Was the model trained on all markers or were multiple models trained on a single marker each? For example, if trained to enhance a single marker (or co-localized group of markers), it could introduce artefacts where it forces signal localization to those areas even for others. What happens if you feed in images with scrambled pixel locations, does it still say the structures are where the training data says they should be? What about markers with different localization from the training set? If you feed those in, does it force them to the location expected by the training data or does it retain their differential true localization and simply enhance the signal?

Reviewer #2 (Public Review):

Summary:
The authors have generated human iPSC cells constitutively expressing the mNG21-10 and tested them by endogenous tagging multiple genes with mNG211 (several tagged iPS cell lines clones were isolated). With this tool, they have explored several weakly expressed cytokinesis genes and gained insights into how cytokinesis occurs.

Strengths:
Human iPSC cells are used.

Weaknesses:
i) The manuscript is extremely incremental, no improvements are present in the split-fluorescent (split-FP) protein variant used nor in the approach for endogenous tagging with split-FPs (both of them are already very well established and used in literature as well as in different cell types).

ii) The fluorescence intensity of the split mNeonGreen appears rather low, for example in Figure 2C the H2BC11, ANLN, SOX2, and TUBB3 signals are very noisy (differences between the structures observed are almost absent). For low-expression targets, this is an important limitation. This is also stated by the authors but image restoration could not be the best solution since a lot of biologically relevant information will be lost anyway.

iii) There is no comparison with other existing split-FP variants, methods, or imaging and it is unclear what the advantages of the system are.

Reviewer #3 (Public Review):

The authors report on the engineering of an induced Pluripotent Stem Cell (iPSC) line that harbours a single copy of a split mNeonGreen, mNG2(1-10). This cell line is subsequently used to take endogenous protein with a smaller part of mNeonGreen, mNG2(11), enabling the complementation of mNG into a fluorescent protein that is then used to visualize the protein. The parental cell is validated and used to construct several iPSC lines with endogenously tagged proteins. These are used to visualize and quantify endogenous protein localisation during mitosis.

I see the advantage of tagging endogenous loci with small fragments, but the complementation strategy has disadvantages that deserve some attention. One potential issue is the level of the mNG2(1-10). Is it clear that the current level is saturating? Based on the data in Figure S3, the expression levels and fluorescence intensity levels show a similar dose-dependency which is reassuring, but not definitive proof that all the mNG2(11)-tagged protein is detected.

Do the authors see a difference in fluorescence intensity for homo- and heterozygous cell lines that have the same protein tagged with mNG2(11)? One would expect two-fold differences, or not?

Related to this, would it be favourable to have a homozygous line for expressing mNG2(1-10)?

The complementation seems to work well for the proteins that are tested. Would this also work for secreted (or other organelle-resident) proteins, for which the mNG2(11) tag is localised in a membrane-enclosed compartment?

The authors present a technological advance and it would be great if others could benefit from this as well by having access to the cell lines.