Biology subjects

Emmanouilidou, A.

Publications and source records attributed to Emmanouilidou, A..

6 recordsLinked to original sources

bioRxiv2025

Accurate characterization of CRISPR-Cas9 genome editing outcomes and mosaicism with near-perfect long reads

BackgroundGenetic mosaicism is a consequence of CRISPR-Cas9 genome editing that is difficult to study, especially when it involves structural variants occurring at low frequency. A comprehensive analysis of mosaicism requires deep and unbiased sequencing of the target loci, with accurate single molecule reads. Here we performed amplification-free PureTarget PacBio sequencing to investigate CRISPR-Cas9 outcomes at on-target and off-target sites in germline edited zebrafish and their offspring. ResultsThirty samples from pooled larvae and individual zebrafish were successfully sequenced, resulting in >1100x average target coverage. The PacBio reads reached an exceptional accuracy (QV39) over the target regions, with every read originating from a unique DNA molecule. The two haplotypes of the target loci displayed a balanced depth of coverage, while long-range PCR of the same samples resulted in skewed data. Further analysis of the PureTarget data revealed widespread genetic mosaicism in individual founder (F0) fish, with up to 18 distinct on-target events and 11 off-target events present in a single adult founder. Several CRISPR-Cas9 editing outcomes, including large structural variants and off-target mutations, were inherited to the F1 generation. Notably, as many as seven unique editing events were found among F1 juvenile offspring from a single founder pair, thereby confirming the presence of genetic mosaicism in germ cells of founder zebrafish. This implies that some consequences of CRISPR-Cas9 editing may emerge only in the second generation. We also analyzed DNA methylation but did not observe altered 5mC CpG signals in genome edited samples. ConclusionsPureTarget enables efficient, accurate and unbiased analysis of the full spectrum of genetic mosaicism in a sample. The potential risks of introducing germ cell mosaicism in founder individuals should be carefully evaluated when designing germline genome editing experiments.

genomics↗

bioRxiv2023

Kita crispants for systematic image-based genetic screens of complex traits in zebrafish larvae

With thousands of loci identified by genome-wide association studies for complex traits, there is a need for in vivo model systems that can reliably and quickly infer the role of large numbers of candidate genes. CRISPR/Cas9-based functional screens in F0 zebrafish represent such a system. However, negative controls used so far - including scrambled guide RNAs (gRNAs), inactivated Cas9, and sham injections - do not elicit the same cellular and organismal responses as mutagenesis by CRISPR/Cas9, and may fuel biased conclusions. Here, we show that targeting kita facilitates efficient optical pre-screening for successful mutagenesis, higher quality imaging data, and efficient classification of cases and controls. We identified and tested two gRNAs that target kita with similarly high mutagenic efficiency and effects on pigmentation, and are free from off-target effects or major effects on cardiometabolic traits. We propose several approaches that will result in valid, unbiased conclusions.

molecular biology↗

bioRxiv2021

CRISPR-Cas9 induces large structural variants at on-target and off-target sites in vivo that segregate across generations

To investigate the extent and distribution of unintended mutations induced by CRISPR-Cas9 in vivo, we edited the genome of fertilized zebrafish eggs and investigated DNA from >1100 larvae, juvenile and adult fish in the F0 and F1 generations. Four guide RNAs (gRNAs) were used, selected from 23 gRNAs with high on-target efficiency in vivo in previous functional experiments. CRISPR-Cas9 outcomes were analyzed by long-read sequencing of on-target sites and off-target sites detected in vitro. In founder larvae, on-target editing of the four gRNAs was 93-97% efficient, and three sites across two gRNAs were identified with in vivo off-target editing. Seven percent of the CRISPR-Cas9 editing outcomes correspond to structural variants (SVs), i.e., insertions and deletions [≥]50 bp. The adult founder fish displayed a mosaic pattern of editing events in somatic and germ cells. The F1 generation contained high levels of genome editing, with all alleles of 46 examined F1 juvenile fish affected by on-target mutations, including four cases of SVs. In addition, 26% of the juvenile F1 fish (n=12) carried off-target mutations. These CRISPR-induced off-target mutations in F1 fish were successfully validated in pooled larvae from the same founder parents. In conclusion, we demonstrate that large SVs and off-target mutations can be introduced in vivo and passed through the germline to the F1 generation. The results have important consequences for the use of CRISPR-Cas9 in clinical applications, where pre-testing for off-target activity and SVs on patient material is advisable to reduce the risk of unanticipated effects with potentially large implications.

genomics↗

bioRxiv2020

Amplification-free long read sequencing reveals unforeseen CRISPR-Cas9 off-target activity

A much-debated concern about CRISPR-Cas9 genome editing is that unspecific guide RNA (gRNA) binding may induce off-target mutations. However, accurate prediction of CRISPR-Cas9 off-target sites and activity is challenging. Here we present SMRT-OTS and Nano-OTS, two amplification-free long-read sequencing protocols for detection of gRNA driven digestion of genomic DNA by Cas9. The methods were assessed using the human cell line HEK293, which was first re-sequenced at 18x coverage using highly accurate (HiFi) SMRT reads to get a detailed view of all on- and off-target binding regions. We then applied SMRT-OTS and Nano-OTS to investigate the specificity of three different gRNAs, resulting in a set of 55 high-confidence gRNA binding sites identified by both methods. Twenty-five (45%) of these sites were not reported by off-target prediction software, either because they contained four or more single nucleotide mismatches or insertion/deletion mismatches, as compared with the human reference. We further discovered that a heterozygous SNP can cause allele-specific gRNA binding. Finally, by performing a de novo genome assembly of the HiFi reads, we were able to re-discover 98.7% of the gRNA binding sites without any prior information about the human reference genome. This suggests that CRISPR-Cas9 off-target sites can be efficiently mapped also in organisms where the genome sequence is unknown. In conclusion, the amplification-free sequencing protocols revealed many gRNA binding sites in vitro that would be difficult to predict based on gRNA sequence alignment to a reference. Nevertheless, it is still unknown whether in vivo off-target editing would occur at these sites.

genomics↗

bioRxiv2018

Zebrafish larvae as a model system for systematic characterization of drugs and genes in dyslipidemia and atherosclerosis

BackgroundHundreds of loci have been robustly associated with circulating lipids, atherosclerosis and coronary artery disease; but for most loci the causal genes and mechanisms remain uncharacterized.\n\nMethodsWe developed a semi-automated experimental pipeline for systematic, quantitative, large-scale characterization of mechanisms, drugs and genes associated with dyslipidemia and atherosclerosis in a zebrafish model system. We validated our pipeline using a dietary (n>2000), drug treatment (n>1000), and genetic intervention (n=384), and used it to characterize three candidate genes in a GWAS-identified pleiotropic locus on chr 19p13.11 (n>500).\n\nResultsOur results show that five days of overfeeding and cholesterol supplementation had independent pro-atherogenic effects, which could be diminished by concomitant treatment with atorvastatin and ezetimibe. CRISPR-Cas9-induced mutations in orthologues of proof-of-concept genes resulted in higher LDL cholesterol levels (apoea), and more early stage atherosclerosis (apobb.1). Finally, our pipeline helped identify putative causal genes for circulating lipids and early-stage atherosclerosis (LPAR2 and GATAD2A).\n\nConclusionsIn summary, our pipeline facilitates systematic, in vivo characterization of drugs and candidate genes to increase our understanding of disease etiology, and can likely help identify novel targets for therapeutic intervention.

genomics↗

bioRxiv2018

Translating GWAS-identified loci for cardiac rhythm and rate using an in vivo, image-based, large-scale genetic screen in zebrafish

A meta-analysis of genome-wide association studies (GWAS) identified eight loci that are associated with heart rate variability (HRV), but candidate genes in these loci remain uncharacterized. We developed an image- and CRISPR/Cas9-based pipeline to systematically characterize candidate genes for HRV in live zebrafish embryos. Nine zebrafish orthologues of six human candidate genes were targeted simultaneously in eggs from fish that transgenically express GFP on smooth muscle cells (Tg[acta2:GFP]), to visualize the beating heart. An automated analysis of repeated 30s recordings of beating atria in 381 live, intact zebrafish embryos at 2 and 5 days post-fertilization highlighted genes that influence HRV (hcn4 and si:dkey-65j6.2 [KIAA1755]); heart rate (rgs6 and hcn4); and the risk of sinoatrial pauses and arrests (hcn4). Exposure to 10 or 25{micro}M ivabradine - an open channel blocker of HCNs - for 24h resulted in a dose-dependent higher HRV and lower heart rate at 5 days post-fertilization. Hence, our screen confirmed the role of established genes for heart rate and rhythm (RGS6 and HCN4); showed that ivabradine reduces heart rate and increases HRV in zebrafish embryos, as it does in humans; and highlighted a novel gene that plays a role in HRV (KIAA1755).

genomics↗