iiinova — where your digital self keeps evolving

Fillip Port @crisprflydesign.bsky.social · 21/09/2026

The planet of the cells. 🔬🧪🔭

Image of a cell culture taken through the ocular of a microscope.

Fillip Port @crisprflydesign.bsky.social · 04/09/2026

Cool holiday moment on the way to the beach. Main BBC News on the radio: "In a scientific breakthrough scientist have mapped all connections in a fly brain." 🐝💪🏼 @jefferis.bsky.social @ribeirocarlitos.bsky.social @mmcosta.bsky.social @mrclmb.ac.uk Finally some pos news. Beach was also great.

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Fillip Port @crisprflydesign.bsky.social · 15/07/2026

Nice paper by the @hietakangaslab.bsky.social using multiplexed Cas12a mutagenesis to show that Salr acts as a negative regulator of growth and lipid storage. link.springer.com/article/10.1...

Figure from the paper showing that Salr CRISPR/Cas12a knockout increases lipid droplet volume in fat body cells.

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Fillip Port @crisprflydesign.bsky.social · 05/06/2026

Our Bachelor student Lara has lot's of talents beyond lab work (unfortunately forgot to take a picture of the amazing cake she made).

Image of a crocheted fruit fly sitting on top of a computer screen.

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Fillip Port @crisprflydesign.bsky.social · 12/05/2026

Interesting new #CRISPR papers. Two studies show that Cas12a2 can act as a RNA-guided DNA shredder to selectively kill cancer cells. www.nature.com/articles/s41... www.biorxiv.org/content/10.6...

Schematic showing the principle of transcript-selective cell killing. Cas12a2 binds specific transcripts via its guideRNA, activating non-specific DNA cleavage in trans, inducing DNA damage and cell death.

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Fillip Port @crisprflydesign.bsky.social · 29/04/2026

Kejelcha broke the 2-hour marathon barrier on Sunday 🤯. He came second and barely made the news. In science, we'd call that getting scooped. But science is not elite sport. It's not a zero-sum game. A "winner takes all" attitude actively discourages a pillar of good science: independent replication.

Image of Yomif Kejelcha, who finished the London marathon in 1:59:41.

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Fillip Port @crisprflydesign.bsky.social · 23/04/2026

Pooled CRISPR screens FTW! A screen in colon carcinoma cells has identified claudin-4 as receptor for a bacterial toxin that is a known cause of colon cancer. www.nature.com/articles/s41...

Figure 1 from the linked paper showing the setup of the CRISPR screen and CLDN4 as the top hit.

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Fillip Port @crisprflydesign.bsky.social · 21/04/2026

Chasing donor backbone insertions in #CRISPR knock-in experiments is a great way to waste an afternoon (or more), unless you use a dominant marker. We like this one from the Carthew lab, in which animals containing the backbone have degenerated eyes: www.tandfonline.com/doi/full/10.... #Drosophila

Image of two fly heads. The left one has normal eyes, while in the right one the eye is degenerated. Eye degeneration is caused by eye specific expression of a RNAi construct against eyes absent (eya), an essential gene for eye morphogenesis. The eya RNAi construct is located on the vector backbone of the donor plasmid that was used in this CRISPR knock-in experiment.

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Fillip Port @crisprflydesign.bsky.social · 13/04/2026

Result of yesterday's sourdough session.

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Fillip Port @crisprflydesign.bsky.social · 31/03/2026

Mapping #CRISPR activity patterns with GRACE. In vivo gene editing sometimes happens at unexpected places. Reporters, such as GRACE, light up the cells that have been mutated. Flies are at @vdrc-flies.bsky.social and plasmids @addgene.bsky.social Described here: www.nature.com/articles/s41...

Figure showing the principle of the GRACE reporter: CRISPR mutagenesis inactivates an uORF that inhibits GFP translation. Below are several examples of Drosophila wing imaginal discs expressing GRACE and Cas12a+ under control of different Gal4 drivers.

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Fillip Port @crisprflydesign.bsky.social · 24/03/2026

Join us in Heidelberg for your PhD! If you are excited about CRISPR genome engineering and would like to develop and apply new methods to investigate Wnt signaling in different model systems, than this project is for you. More info here: my-hbigs.uni-heidelberg.de/site/index.p... 🧪🧬🔬

Header image for the PhD position "Advanced genome engineering to investigate Wnt signaling in animals."

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Fillip Port @crisprflydesign.bsky.social · 29/01/2026

So multiplexed Cas12a is safe and specific, but does it actually improve knockout efficiency? Direct comparisons with two established Cas9 libraries show superior performance, uncovering phenotypes that existing approaches miss.

Part of figure 6 showing the results of a systematic comparison of mutagenesis with Cas12a sgRNA arrays to two currently used Cas9 sgRNA libraries. in both cases multiplexed Cas12a editing outperformed current technology and identified several phenotypes missed by established technology.

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Fillip Port @crisprflydesign.bsky.social · 29/01/2026

The results were very reassuring: >99% of sgRNA arrays showed on-target activity, while we detected no reproducible off-target cutting on the chromosome arms we screened.

Part of figure 5 showing that Cas12a activity is only detected on chromosome arms with sgRNA arrays targeting genes located on this arm and not elsewhere.

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Fillip Port @crisprflydesign.bsky.social · 29/01/2026

Another potential problem: proximity effects, where cutting one gene also deletes neighbors or entire chromosome arms. Testing genes adjacent to characterized loci, we found minimal evidence of such effects in Drosophila (though rare cases do occur).

Part of figure 4 that shows that targeting uncharacterised genes with Cas12a sgRNA arrays does not recapitulate phenotypes known to arise from disruption of neighboring genes (with rare exceptions).

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Fillip Port @crisprflydesign.bsky.social · 29/01/2026

As expected, DNA cutting causes an increase in cell death, which is moderate when cut sites are clustered, but excessive when they are distributed over different chromosomes (which is likely to give rise to genomic rearrangements).

Figure 3 showing the number of apoptotic cells in the wing disc epithelium after clustered or dispersed CRISPR cutting. While clustered cuts are moderately toxic, cuts on different chromosomes are highly detrimental.

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Fillip Port @crisprflydesign.bsky.social · 29/01/2026

We systematically tested Cas12a mutagenesis with 4 sgRNAs per gene in Drosophila. This included building a modular toolkit: sgRNA arrays for 800+ genes, optimized Cas12a transgenes, and activity reporters to map and measure Cas12a activity in vivo.

Part of Figure 2 showing the different tools we build for multiplexed Cas12a mutagenesis in flies.

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Fillip Port @crisprflydesign.bsky.social · 29/01/2026

Make your gene knockouts more efficient with multiplexed Cas12a sgRNAs. Our new paper is out now, with tools available from @addgene.bsky.social , www.plasmids.eu and @vdrc-flies.bsky.social. www.nature.com/articles/s41... #CRISPR #geneediting #Drosophila 🧪🧬✂️🔬🪰 Summary 🧵 below.

Header image with the paper title: "Improved in vivo gene knockout with high specificity using multiplexed Cas12a sgRNAs"

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Fillip Port @crisprflydesign.bsky.social · 06/11/2025

🤯This is wow! Molecular mechanism of mRNA export. by @juliusbrennecke.bsky.social @plaschkalab.bsky.social www.nature.com/articles/s41...

Illustration of the molecular mechanism controlling mRNA export.

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Fillip Port @crisprflydesign.bsky.social · 04/10/2025

High levels of Cas9 are toxic in sensory neurons. Reducing Cas9 levels with uORFs avoids toxicity and is compatible with efficient editing. From @thompsonpeerlab.bsky.social. Fly lines @vdrc-flies.bsky.social #CRISPR #Drosophila www.biorxiv.org/content/10.1...

Figure from the preprint showing sensory neuron morphology with different levels of Cas9 expression.

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Fillip Port @crisprflydesign.bsky.social · 16/07/2025

Hey @nature.com What's up with your share buttons? Can we please also get 🦋 and 🦣 options to not exclusively promote the network of the guy who has done more than most to harm US science?

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Fillip Port @crisprflydesign.bsky.social · 12/07/2025

Some PIs will stop at nothing to motivate their lab members. 🧪

Graffiti at the end of an underpass in Heidelberg saying 'LAB!'.

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Fillip Port @crisprflydesign.bsky.social · 23/06/2025

Journal spam often gets it wrong, but I do agree that "{title}" is a particularly significant study of mine. 😂🙄

Email invitation to a spam journal based on a previous study titled "{title}"

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Fillip Port @crisprflydesign.bsky.social · 15/06/2025

Oh nice. We were there just a few days ago. Did a nice hike up to Lac Bleu between Haudères and Arolla. Nice on a hot day, as most is in the forest.

Picture of Lac Bleu in Valais/Switzerland.

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Fillip Port @crisprflydesign.bsky.social · 27/05/2025

Using #CRISPR to send selected mRNAs to specific subcellular compartments. Exciting technology to probe and harness the spatial organisation of transcripts. www.nature.com/articles/s41...

Screenshot from the linked paper that used RNA-targeting Cas13 to link mRNAs to motor proteins for intracellular transport.

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Fillip Port @crisprflydesign.bsky.social · 26/05/2025

CRISPR mutagenesis is also not perfect. A common limitation is generation of genetic mosaics, where only some cells carry gene knockouts. But our new generation of tools substatially improves on that. Described in the preprint below with flies on their way to VDRC: www.biorxiv.org/content/10.1...

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Fillip Port @crisprflydesign.bsky.social · 26/05/2025

Why use #CRISPR in #Drosophila when RNAi works so well? Because for some genes well is not good enough. Check out new preprint from the lab demonstrating a role for Vps15 in Wingless trafficking. Something that was missed in several RNAi screens. www.biorxiv.org/content/10.1...

Images from the linked preprint showing Wingless stainings in the Drosophila wing imaginal disc. Targeting vps15 with two indepependent RNAi lines (paper also shows the same with a third line) does not result in a phenotype. In contrast, targeting vps15 with two independent sgRNA constructs and Cas9 results in strong accumulation of Wingless protein in perturbed cells.

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Fillip Port @crisprflydesign.bsky.social · 06/03/2025

That's an interesting twist of the "it's not 'fruit fly', it's 'vinegar fly'" debate.

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Fillip Port @crisprflydesign.bsky.social · 15/02/2025

Heidelberg today. Together for freedom, diversity, and democracy.

Pictures from a march against fascism in Heidelberg a week before the German general election.

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Fillip Port @crisprflydesign.bsky.social · 12/01/2025

Sunday beaking: Yoghurt-Emmer Sourdough. #bread

Bread loaf after proofing during the bench rest.

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Fillip Port @crisprflydesign.bsky.social · 10/01/2025

This is such an iconic shot. I suspect the other contestants in this competition did not feel particularly optimistic.

Screenshot from a Guardian article about the World Sports Photography award. Overall winner was an iconic image of the surfer Gabriel Medina at the Olympics.

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Fillip Port @crisprflydesign.bsky.social · 07/01/2025

This is a nice example, because it's know what happens when smo LOF mutations are induced with different efficiency. The currently used Cas9 tools phenocopy induction with medium efficiency, while multiplex Cas12a editing looks exactly like highly efficient induction of known smo LOF clones.

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Fillip Port @crisprflydesign.bsky.social · 07/01/2025

The power of Cas12a-enabled sgRNA multiplexing for gene disruption. Today’s example: smoothened 1/3 #CRISPR #Drosophila

Images of Drosophila wings. Control wings expressing CRISPR components exclusively in the germline have normal morphology. Targeting the central hedgehog signaling component smoothened with Cas9 and either one or two sgRNAs results in wings of reduced size and missing and ectopic vein tissue (middle panels). Targeting smoothened with Cas12a+ and four sgRNAs results in very small wings without veins.

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Fillip Port @crisprflydesign.bsky.social · 17/12/2024

Probably my favorite experiment we ran this year. Screening #CRISPR activity over entire chromosome arms in living animals. From: www.biorxiv.org/content/10.1...

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Fillip Port @crisprflydesign.bsky.social · 03/12/2024

💯 "reinforce the need to investigate genetic outcomes [of CRISPR editing] beyond those accessible to short-read target amplicon next-generation sequencing" from: www.nature.com/articles/s41...

Adaptation of the popular lamppost meme. A person is searching something in the spotlight of a lamppost and is asked: "Is this where your mutations are?" The reply is "No, but this is what I can amplify by PCR." The implication is that amplicon-based methods for mutation detection are missing all mutation outcomes that are incompatible with PCR (e.g. large deletions, translocations, etc.).

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Fillip Port @crisprflydesign.bsky.social · 03/12/2024

Disease mutations can be fixed via #CRISPR induced homology directed repair, but efficiency in human cells is typically low. A drug that increases this efficiency has generated much excitement, but the Corn lab now shows it causes genomic instability. www.nature.com/articles/s41...

Circos plots of CAST-seq-detected chromosomal translocations in edited HSPCs. Addition of the DNA-PKcs inhibitor AZD7648 increases the frequency of translocations.

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Fillip Port @crisprflydesign.bsky.social · 02/12/2024

To confirm that these phenotypes are real we generated additional sgRNA lines. Once you find good lines the phenotype also appears with Cas9-mediated mutagenesis. We also generated germline mutants in trem, which revealed that the gene is essential. 30/n

Heterogenous effects of trem knock-down with RNAi. While RNAi line BL40881 does not result in a wing phenotype (d), knock-down with line KK109779 results in a shortening of the L5 vein and posterior cross vein (e). (f, f’) Mutagenesis with subsequently made sgRNA pairs under control of a ubiquitous U6:3 promoter and act-Cas9 results in lethality with occasional escapers. Escaper animals show loss of posterior veins.

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Fillip Port @crisprflydesign.bsky.social · 02/12/2024

This increased efficiency reveals phenotypes previously missed. Example: The gene trade embargo (trem). Targeting trem with Cas9/HD_CFD does not result in wing phenotypes. But multiplex Cas12a targeting results in penetrant defects in vein differentiation (+ some changes in size and shape). 29/n

Phenotype resulting from wing-specific mutagenesis of trem. Specific loss of posterior veins is observed.

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Fillip Port @crisprflydesign.bsky.social · 02/12/2024

We compared the phenotypic effects of targeting 103 genes for which we also have sgRNA lines in the Cas9 HD_CFD library. The multiplexed Cas12a system induced more severe phenotypes for the majority of wing size regulators. 28/n

Wing size distribution comparing Cas12a+ targeting of known wing morphogenesis genes (orange, n=33) versus control genes (blue, n=18). Clear phenotypic separation demonstrates specificity. See Extended Data Fig. 5 for details. (i) Comparative analysis of wing size phenotypes using Cas12a+/HD12aCFD versus Cas9/HD_CFD systems. Zero values indicate lethal phenotypes. Systems show significantly different effects on tissue size (Wilcoxon signed-rank test, V = 3915, n = 103, p < 0.001, two-tailed).

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Fillip Port @crisprflydesign.bsky.social · 02/12/2024

Gene targeting with Cas12a+ and 4xsgRNA arrays resulted in a clear separation of positive and negative control genes. This is further evidence that this system is specific, well tolerated, and effective. 27/n

Tissue-specific CRISPR screening in wings with Cas12a+ and HD12aCFD sgRNA arrays. (a) Size of individual wings from animals expressing pdm2-Gal4 UAS-uMCas12a+ and HD12aCFD sgRNA arrays targeting the indicated genes. Each dot presents an individual wing (with typically between 10 - 20 wings measured per genotype) and the black rectangle represents the mean. Selected lines targeting genes known to affect or not affect wing morphogenesis are colored in orange or blue according to the color scheme in (b). While sgRNA targeting negative control genes have minimal effects on wing size, sgRNAs targeting positive control lead to a clear reduction in wing size. Phenotypes that were lethal are classified as zero and visible towards the right of the graph. (b) List of genes considered as positive (orange rectangle) or negative (blue rectangle) controls based on previous literature. Wing size of these lines is also plotted in Figure 3h.

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Fillip Port @crisprflydesign.bsky.social · 02/12/2024

For a quantitative comparison of mutagenesis effects across many genes, we measured the organ size of the adult wing as our phenotypic readout. 26/n

Schematic of the experimental workflow. Tissue-specific CRISPR results in wing specific mutagenesis. Wing size is analyzed with help of a custom Cellpose model.

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Fillip Port @crisprflydesign.bsky.social · 02/12/2024

Another example: Mutagenesis of the Hedgehog signaling component smoothened. The multiplexed Cas12a system leads to much more efficient gene disruption. Also note the stronger reduction in size of the mutated area – Smo is important for tissue growth. 24/n

Immunofluorescent staining of Smo in wing imaginal discs. Conditional mutagenesis in the pouch region (dotted line) with Cas12a+ and HD12aCFD sgRNAs results in more effective gene knock-out. Note correlation between knock-out efficiency and pouch size. Scale bar: 50 µm. (f) Quantification of Smo protein loss. Data points show affected area per disc, with mean (columns) and standard deviation (error bars). ***p<0.001, independent two-sample t-test.

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Fillip Port @crisprflydesign.bsky.social · 02/12/2024

Here is a comparison for disrupting the white gene in the eye. Remember the visual in the first post of this thread? It was based on this experiment. 23/n

Fig. 3a,b: The HD12aCFD system improves disruption of white (w) in the eye. Representative images of eyes from flies with no sgRNA (control) or Cas9 and a single sgRNA or Cas12a+ and a HD12aCFD array. All genotypes contain three functional copies of w due to transgene markers. (b) Quantification of white eye area. Individual data points show white area per eye, with mean (columns) and standard deviation (error bars). **p<0.001, independent two-sample t-test.

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Fillip Port @crisprflydesign.bsky.social · 02/12/2024

And this is the result: Complete separation between lines targeting 2L between fluorophore and centromer (all show strong, reproducible LOH) and lines targeting genes elsewhere in the genome (none show reproducible LOH). 19/n

Fig. 2g: High-throughput screening for LOH on chromosome arm 2L reveals high on-target and undetectable off-target activity. 8197 wing discs expressing Cas12a+, GFP encoded on 2L and one of 525 HD12aCFD sgRNA arrays were screened for LOH. All 82 HD12aCFD lines targeting 2L between GFP and the centromere resulted in LOH, demonstrating robust on-target activity. Lines targeting 2L without LOH phenotype had target sites distal to GFP. Importantly, 443 HD12aCFD lines targeting other chromosome arms showed no reproducible LOH, indicating absence of detectable off-target activity on 2L (16% of the Drosophila genome). Sporadic low-level LOH events were not reproducible (Extended Data Fig. 2) and likely represent background rates.

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Fillip Port @crisprflydesign.bsky.social · 02/12/2024

So we built a system where we can observe Cas12a-induced LOH in living larvae and conducted a screen of 2197 sgRNAs clustered in 525 arrays. Our reporter is inserted on the tip of chromosome 2L, hence the screen reports on CRISPR activity over 21 megabases (~16% of the genome). 18/n

Fig. 2e: A system for high-throughput LOH screening in vivo. The GMR11F02 enhancer allows highly selective expression of GFP in the pouch region of wing and haltere imaginal discs. (f) Control wing disc showing uniform GFP expression throughout wing pouch cells, with natural variations in expression level. (g) Example of LOH in a wing disc of a living larva. LOH events are readily detected as clearly defined patches lacking GFP fluorescence. Scale bar: 50 µm.

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