Fillip Port

@crisprflydesign.bsky.social

2431 Followers 957 Following

Scientist at the German Cancer Reseach Center in Heidelberg. We develop genome engineering tools for functional genomics. #CRISPR #Drosophila #openscience

Figure from the paper showing that Salr CRISPR/Cas12a knockout increases lipid droplet volume in fat body cells.
Image of a crocheted fruit fly sitting on top of a computer screen.
Hiking shoes in front of peaks of the Italian Dolomites.
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.
Image of Yomif Kejelcha, who finished the London marathon in 1:59:41.
Figure 1 from the linked paper showing the setup of the CRISPR screen and CLDN4 as the top hit.
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.
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.
Header image for the PhD position "Advanced genome engineering to investigate Wnt signaling in animals."
Drosophila wing imaginal disc stained with DSHB antibody 4D4 (anti-wingless) in red and Hoechst in blue.
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.
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.
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).
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.
Part of Figure 2 showing the different tools we build for multiplexed Cas12a mutagenesis in flies.
Header image with the paper title: "Improved in vivo gene knockout with high specificity using multiplexed Cas12a sgRNAs"
Illustration of the molecular mechanism controlling mRNA export.
Figure from the preprint showing sensory neuron morphology with different levels of Cas9 expression.
Graffiti at the end of an underpass in Heidelberg saying 'LAB!'.
Email invitation to a spam journal based on a previous study titled "{title}"
F1 playing guitar at a school band gig.
Picture of Lac Bleu in Valais/Switzerland.
Screenshot from the linked paper that used RNA-targeting Cas13 to link mRNAs to motor proteins for intracellular transport.
Visualization of the main improvement of the HD12aCFD sgRNA library on gene knockout in multicellular tissues: While previous sgRNA libraries often generated genetic mosaics where many cells retained gene function, the HD12aCFD library uses higher order sgRNA multiplexing to create mosaics with a high fraction of knockout cells.
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.
Picture of a front yard garden. The earth is mostly bare with only a few shrubs visible.
Pictures from a march against fascism in Heidelberg a week before the German general election.
Bread loaf after proofing during the bench rest.
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.
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.
Measuring CRISPR activity over entire chromosome arms by visualizing loss of heterozygosity (LOH). In the first graph the screened sgRNA lines are plotted chronologically (in the order we screened them), revealing little structure in the data. In the second graph the sgRNAs are ordered according to the genomic position of their target site. This revealed that only lines that target chromosome 2L (N=82) give rise to LOH, while none of the ones with target sites elsewhere does (N=443). Overall, this indicates that the CRISPR system described in the linked preprint is highly robust and specific.
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.).
Circos plots of CAST-seq-detected chromosomal translocations in edited HSPCs. Addition of the DNA-PKcs inhibitor AZD7648 increases the frequency of translocations.
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.
Phenotype resulting from wing-specific mutagenesis of trem. Specific loss of posterior veins is observed.
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).
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.
Schematic of the experimental workflow. Tissue-specific CRISPR results in wing specific mutagenesis. Wing size is analyzed with help of a custom Cellpose model.
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.
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.
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.
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.
Fig. 2b: CRISPR-induced LOH in larval wing imaginal discs. Tissues express Cas12a+, sgRNA arrays, and have a heterozygous mScarlet (mScar) transgene inserted on chromosome arm 2L. Left panel: sgRNAs targeting a different chromosome arm (3R) do not result in LOH. Right panel: sgRNAs targeting a locus between the mScar transgene and the centromere result in frequent LOH resulting in loss or gain of mScar expression. Scale bar: 50 µm.
Fig. 2a: Schematic illustrating mechanisms of loss of heterozygosity (LOH). Crossover between homologous chromosomes results in mitotic recombination and copy number neutral (CN) LOH, leading to homozygous daughter cells (+/+ or -/-) from heterozygous parent cells (+/-). Large chromosomal deletions or chromotrypsis lead to copy number loss (CL) LOH.
Fig. 1g:Quantification of clDCP1-positive apoptotic area in the pdm2-Gal4 expression domain. Points are individual samples, columns represent the mean and error bars the standard deviation. There is no significant (n.s.) difference whether gene targeting is performed with two sgRNAs and Cas9 or four sgRNA and Cas12a+ (Welch Two Sample T-Test)
Fig. 1g:Quantification of clDCP1-positive apoptotic area in the pdm2-Gal4 expression domain. Points are individual samples, columns represent the mean and error bars the standard deviation. There is no significant (n.s.) difference whether gene targeting is performed with two sgRNAs and Cas9 or four sgRNA and Cas12a+ (Welch Two Sample T-Test).
Schematic of 4x sgRNA arrays for Cas9 or Cas12a. Cas12a arrays are significantly smaller in size.
Fig.1c: Individual alleles of a multi-cut reporter were isolated by germline transmission and identified by sequencing. Multiplexing of target sites results in frequent deletions between target sites.
Schematic of a CRISPR-induced genetic mosaic. A substantial fraction of cells retains gene expression.
Schematic of a prototypical CRISPR gene disruption experiment. Cas9 mediates a double strand break, which results in mutations through error prone DNA repair.
Animation of the main advance of the study. Current CRISPR systems often result in incomplete gene knock-out in tissues (cells that retain expression of the blue protein). Targeting the same gene with several sgRNAs results in a strong increase in knockout efficiency.
Screenshot from the showyourstripes website showing yearly average temperature in Frankfurt/Germany.
Figure from the linked paper showing that recruitment of Cas9 to nascent RNA via MS2 or boxB increases targeting of nearby cut sites.
Two Drosophila larva. The upper one has a very large hyperplasia in the head region.