PREreview del Chromosome-Specific Aneuploidy Engineering via dCas9-Induced Centromeric Chromatin Relaxation

Review coordinated via ASAPbio’s crowd preprint review

This review reflects comments and contributions by Joseph Biggane and Andreia Pereira. Review synthesized by Joseph Biggane.

This study investigated a novel method for manipulating ploidy in human immortalized, primary, and induced pluripotent stem cell lines. Centromere-specific gRNAs and dCas9 were used to interfere with kinetochore assembly and produce aneuploidy in targeted chromosomes of dividing cells.  The authors presented evidence for the efficiency and specificity of aneuploidy induction by this method.  Additional evidence suggested that aneuploidy of whole or partial chromosomes was induced via kinetochore disruption.  This method provides a novel means to reliably generate model cell lines for the study of the mechanisms and downstream consequences of aneuploidies.

Positive aspects of this study:

• This study addresses a unique problem.  The mechanisms and implications of partial and whole-chromosome aneuploidies are poorly understood.  This study provides a foundation for future researchers to better understand not only developmental abnormalities but also diseases where aneuploidy is common, such as cancers.

• The implementation of immortalized, primary, and induced pluripotent stem cells demonstrated a fantastic degree of robustness for this method.

• Several aspects of this study that were questioned by the reviewers early in the study were addressed later in the preprint.  This provided support and confidence in the conclusions made by the authors.

Crowd Review Comments:

• Regarding the Abstract:

  • In the opening sentence, the phrasing does not account for other types of chromosomal abnormalities, which would not be considered aneuploidy.

• Regarding the Introduction:

  • It would be helpful if the authors could add information about what readers should expect when observing aneuploidy events in somatic cells, resulting from mitosis, instead of in germ-line, resulting from meiosis.  While it's nuanced, there are some differences.  Also, it would be helpful to discuss acquired uniparental disomy and loss of heterozygosity, especially given the VHL emphasis later in the paper.

  • The third and fourth paragraphs stuck out to the reviewers as a very well-documented “state-of-the-field” description.

• Regarding the Results:

  • In subsection ‘Centromeric dCas9 recruitment induces efficient chromosome-specific mis-segregation’:

    • In reference to “...we unexpectedly observed that this approach also induced efficient Chr3-specific mis-segregation and micronucleus formation (Fig. 1a)”, the reliance on micronucleus formation as a measure of success was a major unanswered question for the reviewers.  From these data, it is difficult to discern if the aneuploidy would be functionally significant for the daughter cell that gained the extra chromosome.  The isolation of the extra chromosome in an extrachromosomal region caused the reviewers to question whether this type of aneuploidy would persist, be duplicated, etc.

• In reference to “We confirmed chromosome-specific micronucleus induction across different models for the selected 23 gRNAs (Fig. 1c,d, and Extended Data Fig. 4)...”, the authors used FISH to validate that the gRNA is identifying the correct chromosome in extended figure 4;  However, even after going back to the high-resolution figure posted on bioRxiv, and zooming way in, it was very difficult to see the FISH probe fluorescence in many of the cases.  It would be helpful if the authors could provide some higher magnification images.  Additionally, some of the DAPI and FISH images are rotated out of alignment.

• In subsection ‘Kinetochore chromatin relaxation underlies dCas9-induced kinetochore rupture and chromosome mis-segregation’:

  • In reference to “Consistently, following mitotic exit, only Chr3 in interphase micronuclei presented a near-complete loss of the inner kinetochore (Fig. 2c).”, in Figure 2C, Panel 2, there seems to be clear CREST fluorescence in at least the furthest left micronucleus, which seems to contradict this statement.

• In reference to “Overall, our data suggest that CRISPRt induces kinetochore rupture and chromosome mis-segregation via dCas9-induced relaxation of kinetochore chromatin (Fig. 2f).”, the reviewers were somewhat skeptical of the kinetochore rupture conclusion, but the relaxation ATAC-seq data seemed pretty interesting/promising.

• In subsection ‘CRISPRt induces whole chromosome aneuploidy and arm-level aneuploidy with centromeric breakage’:

  • In reference to “We confirmed that CRISPRt-induced mis-segregation resulted in both whole chromosome gains and losses (Fig. 3a)”, the reviewers considered if there are other possibilities that should be discussed as well.  Since these are nondisjunctions occurring during mitosis, and both chromosomes are being manipulated, there should at least be a possibility for a double nondisjunction, leading to tetraploid and null daughter cells.

• In subsection ‘CRISPRt generates Chr13, 21, X or Y aneuploidy in iPSCs’:

  • In reference to “To test CRISPRt for targeted aneuploidy manipulation in iPSCs, we applied Chr13/21, X or Y CRISPRt in euploid iFCI013 iPSC line, followed by single-cell chromosome copy number characterisation using single-cell RNA sequencing (scRNA-seq) and InferCNV analyses (Fig. 4a, see Methods)”, the reviewers suggested that these methods could provide a means to test for uniparental disomy emergence.  It might be interesting and insightful, given that uniparental disomy-induced copy-neutral loss of heterozygosity is a known phenomenon in several cancers.

• In subsection ‘CRISPRt generates ccRCC initiating Chr3 aneuploid event in renal epithelial cells’:

  • In reference to “In addition, we performed VHL WT-allele-specific knockout (VHL-ASK) to recapitulate the somatic inactivation of the remaining VHL WT copy (Fig. 5a and Extended Data Fig. 9a,b).”, the reviewers appreciated the methods section describing how this was achieved.

• Regarding figures:

  • Figure 1:

    • In this figure legend, for 1b, the authors should consider adding an explanation for why 5% is used as the threshold for successful gRNA function.

  • Figure 2:

    • There is a significant mismatch between the background fluorescence seen in the CREST images from Figure 2a/b when compared to Figure 2c.  This is pretty troubling to me, given that they are using an absence of CREST signaling to make a conclusion about Figure 2c.

• The reviewers questioned why the DAPI fluorescence was always visible.  While it might be useful to see the chromatin, the DAPI fluorescence is obscuring the fluorescence in the other channels. In conventional fluorescence imaging, DAPI would have its own channel image, and then all channels are overlaid in a merged image.  Perhaps, the authors might consider using an outline from the DAPI channel overlaid on the other fluorophore channels

  • The oversaturation of the background could probably be avoided without the DAPI competition as well.

• Figure 3:

  • In Figure 3A, this image seems to show evidence of the chromosome gains being integrated into the nucleus, but the authors might consider addressing the gap between micronucleus integration and integration within the nucleus.

• Figure 4:

  • In Figures 4 b, c, the reviewers suggest that it would have been beneficial to remain consistent and show karyotype images along with the copy number analysis.  

Conflicts of interest:

• None declared.

Competing interests

The authors declare that they have no competing interests.