Anna Zofia Komisarczuk

• Labun, Kornel; Rio, Oline Eriksen; Dahal-Koirala, Shiva; Komisarczuk, Anna Zofia; Valen, Eivind & Haapaniemi, Emma Maria (2026). SNIPSNP: precision design of CRISPR/Cas9 knock-in reagents for variant correction and disease modeling. Nucleic Acids Research (NAR). ISSN 0305-1048. 54(W1), s. W145–W153. doi: 10.1093/nar/gkag409. Fulltekst i vitenarkiv Vis sammendrag

  We present SNIPSNP (crisprtools.org/snipsnp), a comprehensive bioinformatics pipeline for designing experiments for CRISPR-induced homology-directed repair (HDR). The tool addresses the critical challenge of Cas9 re-cleavage by simplifying the selection of “blocking” silent variants that are effective at inhibiting RNP binding upon donor-templated editing. SNIPSNP handles complex edits, including indels, and uses multi-objective optimization to balance editing efficiency with biological safety. From user-defined wild-type and desired HDR alleles, the pipeline identifies candidate guides, annotating them with integrated efficiency scores and genome-wide off-target assessments. Uniquely, SNIPSNP evaluates guide binding against the post-edit genome to determine whether the therapeutic variant alone disrupts repeated Cas9 recognition. When necessary, it introduces synonymous blocking variants, prioritizing PAM and seed regions to minimize re-cleavage probability and editing of the wild-type (WT) allele when editing heterozygous variants. All candidate modifications undergo safety profiling and prioritization of known benign variants from dbSNP. We experimentally validated SNIPSNP and benchmarked it on pathogenic inborn error of immunity variants in primary patient T-cells. Across loci, SNIPSNP-designed templates outperform standard “correction-only” strategies, demonstrating enhanced precision editing, and reduced re-cleavage, establishing SNIPSNP as a robust platform for genome editing and disease modeling.

• Araya, Robin Aasegg; Reinar, William Brynildsen; Tørresen, Ole K; Goubert, Clément; Daughton, Tara Jane & Hoff, Siv Nam Khang [Vis alle 12 forfattere av denne artikkelen] (2025). Chromosomal Inversions Mediated by Tandem Insertions of Transposable Elements. Genome Biology and Evolution (GBE). 17(8). doi: 10.1093/gbe/evaf131. Fulltekst i vitenarkiv Vis sammendrag

  Chromosomal inversions play a crucial role in evolution by influencing phenotypes through the linkage of coadapted alleles. While inversions have been found across a large number of taxa, mapping and characterizing inversion breakpoint regions remain challenging, often due to the presence of complex tandem repeats and transposable elements. Here, we identify and quantify transposable elements in the breakpoints of the four large-scale inversions previously reported in Atlantic cod, leveraging on three high-quality long-read-based reference genome assemblies for the Norwegian Coastal cod, the Northeast Arctic cod, and Celtic cod ecotypes. We detected a significant enrichment of transposable element orders and superfamilies with terminal inverted repeats within the inversion breakpoint regions of chromosomes 1, 7 and 12. Notably, we discovered a tandem accumulation of miniature inverted-repeat transposable elements belonging to a family of hAT transposons, exclusively residing in the breakpoints of the inverted haplotype on chromosomes 1 and 7 found in the Northeast Arctic cod. The accumulation of tandemly arranged transposable elements with high sequence similarity in breakpoint regions suggests that they have driven the appearance of inversions through ectopic recombination, further supporting the potential of transposable elements in facilitating chromosomal reorganizations with large evolutionary implications.

• Labun, Kornel; Rio, Oline Eriksen; Tjeldnes, Håkon; Swirski, Michał; Komisarczuk, Anna Zofia & Haapaniemi, Emma Maria [Vis alle 7 forfattere av denne artikkelen] (2025). CHOPOFF: symbolic alignments enable fast and sensitive CRISPR off-target detection. bioRxiv - the preprint server for biology. doi: 10.1101/2025.01.06.603201. Fulltekst i vitenarkiv Vis sammendrag

  ABSTRACT CRISPR/Cas systems offer powerful tools for genome editing, but their therapeutic application is hampered by the risk of unintended off-target mutations. Many molecular methods have been established to detect off-target editing, however, their sensitivity depends on first identifying potential sites using in silico methods. However, these in silico prediction methods are challenged by a trade-off between speed and sensitivity, and can fail to comprehensively detect all edited off-target sites. Here, we demonstrate that ignoring bulges has led to missing editing at off-target sites in previous studies and that continuing this practice can lead to inflated claims of fidelity. As a solution, we introduce the concept of symbolic alignments to efficiently identify off-targets without sacrificing sensitivity. We further present specialized data structures that enable rapid, alignment-free probabilistic ranking of guide RNAs based on their predicted off-target burden. Implemented in the tool CHOPOFF, these innovations accommodate mismatches, bulges (insertions/deletions), and incorporate genomic sequence variants for personalized off-target assessment. Benchmarking demonstrates that CHOPOFF significantly outperforms state-of-the-art tools in both prediction accuracy and computational speed. Availability CHOPOFF command line available at https://github.com/JokingHero/CHOPOFF.jl CHOPOFF web server available at https://crisprtools.org/chopoff

• Kopcil, Pavel; Ervik, Carolina W.; Reint, Ganna; Mamia, Katariina; Szymanska, Monika & Dahal-Koirala, Shiva [Vis alle 17 forfattere av denne artikkelen] (2025). Editing of ADA2 Point Mutation in Human Hematopoietic Stem Cells. bioRxiv (preprint article). doi: 10.1101/2025.08.14.670303. Fulltekst i vitenarkiv Vis sammendrag

  Abstract Background The homozygous ADA2 : c.506G>A (p.Arg169Gln; p.R169Q) variant accounts for majority of Deficiency in Adenosine Deaminase 2 (DADA2). This monogenic disorder may be amenable to ex vivo gene therapy by correcting the pathogenic mutation in CD34+ hematopoietic stem and progenitor cells (HSPCs). Objective To apply CRISPR-Cas9 and homology-directed repair (HDR) as a surrogate strategy to model correction of the pathogenic ADA2 c.506G>A variant in healthy cord blood HSPCs. Methods HSPCs were electroporated with optimised CRISPR-Cas9 editing reagents, and editing outcomes, including HDR and on-target deletions, were quantified by ddPCR. Cell functionality was assessed through colony-forming unit (CFU) assays and by xenotransplantation into NOD SCID Gamma (NSG) mice. Two HDR enhancement strategies were tested: (1) genetic inhibitors of p53 and non-homologous end joining (NHEJ) pathways, and (2) pharmacological NHEJ inhibition. Results Small-molecule NHEJ inhibitors increased HDR efficiency approximately two-fold (from ∼40 % to ∼80 %). Edited HSPCs retained normal CFU capacity and successfully engrafted in NSG mice. However, up to 8 % of edited cells exhibited on-target chromosome loss, though this declined over time. Up to 40 % of T cells and fibroblasts demonstrated similar losses under NHEJ inhibitors treatment. In contrast, genetically encoded inhibitors did not improve HDR. Conclusion The ADA2 p. c.506G>A variant can be effectively edited employing surrogate strategy in HSPCs without impairing functionality. Although pharmacological inhibition of NHEJ enhances HDR efficiency, it also increases the risk of on-target chromosome aberrations, highlighting the need for careful consideration of the associated risks and benefits in therapeutic gene editing. Key messages 1) The ADA2 p.R169Q variant can be efficiently corrected via HDR, and the edited CD34+ HSPCs retain their engraftment capability in NSG mice. 2) Pharmacological inhibition of NHEJ using small-molecule inhibitors increases HDR efficiency but is associated with significant on-target deletions and chromosomal arm loss, particularly in differentiated cell types, and in a donor-dependent manner. Capsule summary The ADA2 p.R169Q variant is a viable target for precision gene editing in hematopoietic stem cells. Although inhibition of NHEJ improves HDR efficiency, it concomitantly increases the risk of large on-target deletions, particularly in differentiated cells.

• Mamia, Katariina Aino Inkeri; Kolbeinsdottir, Solrun; Labun, Kornel; Li, Zhuokun; Komisarczuk, Anna Zofia & Keskitalo, Salla [Vis alle 32 forfattere av denne artikkelen] (2025). Precision T cell correction platform for Inborn Errors of Immunity. Molecular Therapy. ISSN 1525-0016. 33(11), s. 5624–5643. doi: 10.1016/j.ymthe.2025.08.018. Fulltekst i vitenarkiv Vis sammendrag

  CRISPR/Cas9 gene editing is a promising tool to correct pathogenic variants for autologous cell therapies targeting Inborn errors of immunity (IEI). Current strategies, such as gene knockout or cDNA knock-in, address many single-gene defects but can disrupt gene expression, highlighting the need for precise correction platforms. While transplanting corrected autologous hematopoietic stem cells is a curative approach, it is unsuitable for patients with advanced disease, inflammation or acute infections. As correcting T cells is an alternative therapeutic strategy for lymphoid IEIs, we present an efficient T cell single nucleotide variant (SNV) correction platform based on homology-directed repair (HDR). By using STAT1 gain-of-function (STAT1-GOF), Cartilage Hair Hypoplasia (CHH), Deficiency of ADA2 (DADA2), and Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) as IEI models, we demonstrate that our platform achieves up to 80% correction, with resultant functional correction of the disease phenotype in the selected models. Furthermore, we performed safety profiling using GUIDE-seq, single-cell RNA sequencing, long-read genome sequencing, and proteomics analysis and detected no genomic, transcriptomic, or proteomic aberrations. This study establishes HDR-based SNV editing as a portable method for developing clinical autologous T cell therapies and represents a promising step toward a broad-spectrum gene correction platform for treating diverse monogenic immune disorders.

• Ciani, Elia; Stormoen, Marit; Antonsen, Stein Ivar; Nilsen, Frank; Jørgensen, Even & Komisarczuk, Anna Zofia (2025). Optimization of calcium oxide treatment against salmon louse (Lepeophtheirus salmonis). A controlled laboratory study. Aquaculture Reports. 43. doi: 10.1016/j.aqrep.2025.102894. Fulltekst i vitenarkiv
• Borchel, Andreas; Komisarczuk, Anna Zofia & Nilsen, Frank (2022). Sex differences in the early life stages of the salmon louse Lepeophtheirus salmonis (Copepoda: Caligidae). PLOS ONE. 17(3). doi: 10.1371/journal.pone.0266022. Fulltekst i vitenarkiv Vis sammendrag

  Salmon lice are ectoparasites on salmonids and feed on blood, mucus, and skin from their hosts. This causes high annual costs for treatment and control for the aquaculture industry. Salmon lice have a life cycle consisting of eight life stages. Sex determination by eye is only possible from the sixth stage onwards. A molecular sex determination has not been carried out so far, even though few individual sex-linked SNPs have been reported. In the present study, we used known sex-specific SNPs as a basis to sequence the complete sex-specific gene variants and used the sequence information to develop a sex determination assay. This assay could be used to determine the developmental speed of the two sexes already in the earliest life stages. Additionally, we sampled salmon lice in the nauplius II stage, determined the sex of each individual, pooled their RNA according to their sex, and used RNA sequencing to search for differences in gene expression and further sex-specific SNPs. We succeeded in developing a sex-determination assay that works on DNA or RNA from even the earliest larval stages of the salmon louse after hatching. At these early developmental stages, male salmon lice develop slightly quicker than females. We detected several previously unknown, sex-specific SNPs in our RNA-data seq, but only very few genes showed a differential expression between the sexes. Potential connections between SNPs, gene expression, and development are discussed.

• Skern-Mauritzen, Rasmus; Malde, Ketil; Eichner, Christiane; Dondrup, Michael; Furmanek, Tomasz & Besnier, Francois [Vis alle 23 forfattere av denne artikkelen] (2021). The salmon louse genome: Copepod features and parasitic adaptations. Genomics. ISSN 0888-7543. 113(6), s. 3666–3680. doi: 10.1016/j.ygeno.2021.08.002. Fulltekst i vitenarkiv Vis sammendrag

  Copepods encompass numerous ecological roles including parasites, detrivores and phytoplankton grazers. Nonetheless, copepod genome assemblies remain scarce. Lepeophtheirus salmonis is an economically and ecologically important ectoparasitic copepod found on salmonid fish. We present the 695.4 Mbp L. salmonis genome assembly containing ≈60% repetitive regions and 13,081 annotated protein-coding genes. The genome comprises 14 autosomes and a ZZ-ZW sex chromosome system. Assembly assessment identified 92.4% of the expected arthropod genes. Transcriptomics supported annotation and indicated a marked shift in gene expression after host attachment, including apparent downregulation of genes related to circadian rhythm coinciding with abandoning diurnal migration. The genome shows evolutionary signatures including loss of genes needed for peroxisome biogenesis, presence of numerous FNII domains, and an incomplete heme homeostasis pathway suggesting heme proteins to be obtained from the host. Despite repeated development of resistance against chemical treatments L. salmonis exhibits low numbers of many genes involved in detoxification.

• Komisarczuk, Anna Zofia; Kongshaug, Heidi; Li, Ming & Nilsen, Frank (2019). RNAi mediated myosuppressin deficiency affects muscle development and survival in the salmon louse (Lepeophtheirus salmonis). Scientific Reports. 9(1). doi: 10.1038/s41598-019-43515-w. Fulltekst i vitenarkiv Vis sammendrag

  Muscle activity is regulated by stimulatory and inhibitory neuropeptides allowing for contraction and relaxation. In Arthropods, one of the important myoinhibitors is Myosuppressin, belonging to FMRFamide-like peptides, that was shown to have inhibitory effects on visceral muscle contraction and to regulate vital physiological processes including reproduction or feeding. We have identified myosuppressin in salmon louse Lepeophtheirus salmonis (LsalMS) and systematically characterised its function and complex abnormalities emerging after LsalMS knockdown by RNAi in all developmental stages in this species. Immunohistochemistry analysis localized the LsalMS mainly to the central nervous system, but also to the vital organs within the alimentary tract and the reproductive system. The most striking feature of LsalMS deficiency during lice development was severe reduction of the muscle content, with abnormalities detected in both the visceral and skeletal muscles. Moreover, down-regulation of LsalMS affects moulting, spermatophore deposition and feeding by affecting development of the intestinal wall and increasing its contraction frequency.

• Komisarczuk, Anna Zofia; Kongshaug, Heidi & Nilsen, Frank (2018). Gene silencing reveals multiple functions of Na+/K+-ATPase in the salmon louse (Lepeophtheirus salmonis). Experimental parasitology. ISSN 0014-4894. 185, s. 79–91. doi: 10.1016/j.exppara.2018.01.005. Fulltekst i vitenarkiv
• Komisarczuk, Anna Zofia; Grotmol, Sindre & Nilsen, Frank (2017). Ionotropic receptors signal host recognition in the salmon louse (Lepeophtheirus salmonis, Copepoda). PLOS ONE. doi: 10.1371/journal.pone.0178812. Fulltekst i vitenarkiv
• Borchel, Andreas; Komisarczuk, Anna Zofia; Rebl, Alexander; Goldammer, Tom & Nilsen, Frank (2017). Systematic identification and characterization of stress-inducible heat shock proteins (HSPs) in the salmon louse (Lepeophtheirus salmonis). Cell stress & chaperones (Print). ISSN 1355-8145. 23(1), s. 127–139. doi: 10.1007/s12192-017-0830-9. Fulltekst i vitenarkiv
• Soulika, Marina; Kaushik, Anna-Lila; Mathieu, Benjamin; Lourenço, Raquel; Komisarczuk, Anna Zofia & Romano, Sebastian Alejo [Vis alle 13 forfattere av denne artikkelen] (2016). Diversity in cell motility reveals the dynamic nature of the formation of zebrafish taste sensory organs. Development. ISSN 0950-1991. 143(11), s. 2012–2024. doi: 10.1242/dev.134817. Fulltekst i vitenarkiv
• Sundström, Elisabeth; Komisarczuk, Anna Zofia; Jiang, Lin; Golovko, Anna; Navratilova, Pavla & Rinkwitz, Silke [Vis alle 8 forfattere av denne artikkelen] (2012). Identification of a melanocyte-specific, microphthalmia-associated transcription factor-dependent regulatory element in the intronic duplication causing hair greying and melanoma in horses. Pigment Cell & Melanoma Research. ISSN 1755-1471. 25(1), s. 28–36. doi: 10.1111/j.1755-148X.2011.00902.x. Fulltekst i vitenarkiv
• Amsterdam, Adam; Lai, Kevin; Komisarczuk, Anna Zofia; Becker, Thomas S.; Bronson, Roderick T & Hopkins, Nancy [Vis alle 7 forfattere av denne artikkelen] (2009). Zebrafish Hagoromo Mutants Up-Regulate fgf8 Postembryonically and Develop Neuroblastoma. Molecular Cancer Research. ISSN 1541-7786. 7(6), s. 841–850. doi: 10.1158/1541-7786.MCR-08-0555. Fulltekst i vitenarkiv
• Komisarczuk, Anna Zofia; Kawakami, Koichi & Becker, Thomas S. (2009). Cis-regulation and chromosomal rearrangement of the fgf8 locus after the teleost/tetrapod split. Developmental Biology. ISSN 0012-1606. 336(2), s. 301–312. doi: 10.1016/j.ydbio.2009.09.029. Fulltekst i vitenarkiv
• Topp, Stefanie; Stigloher, Christian; Komisarczuk, Anna Zofia; Adolf, Birgit; Becker, Thomas S. & Bally-Cuif, Laure (2008). Fgf signaling in the zebrafish adult brain: Association of Fgf activity with ventricular zones but not cell proliferation. Journal of Comparative Neurology. ISSN 0021-9967. 510(4), s. 422–439. doi: 10.1002/cne.21802. Fulltekst i vitenarkiv
• Kikuta, Hiroshi; Laplante, Mary; Navratilova, Pavla; Komisarczuk, Anna Zofia; Engstrøm, Pær & Fredman, David [Vis alle 22 forfattere av denne artikkelen] (2007). Genomic regulatory blocks encompass multiple neighboring genes and maintain conserved synteny in vertebrates. Genome Research. ISSN 1088-9051. 17(6), s. 545–555. doi: 10.1101/gr.6086307. Fulltekst i vitenarkiv Vis sammendrag

  We report evidence for a mechanism for the maintenance of long-range conserved synteny across vertebrate genomes. We found the largest mammal-teleost conserved chromosomal segments to be spanned by highly conserved noncoding elements (HCNEs), their developmental regulatory target genes, and phylogenetically and functionally unrelated "bystander" genes. Bystander genes are not specifically under the control of the regulatory elements that drive the target genes and are expressed in patterns that are different from those of the target genes. Reporter insertions distal to zebrafish developmental regulatory genes pax6.1/2, rx3, id1, and fgf8 and miRNA genes mirn9-1 and mirn9-5 recapitulate the expression patterns of these genes even if located inside or beyond bystander genes, suggesting that the regulatory domain of a developmental regulatory gene can extend into and beyond adjacent transcriptional units. We termed these chromosomal segments genomic regulatory blocks (GRBs). After whole genome duplication in teleosts, GRBs, including HCNEs and target genes, were often maintained in both copies, while bystander genes were typically lost from one GRB, strongly suggesting that evolutionary pressure acts to keep the single-copy GRBs of higher vertebrates intact. We show that loss of bystander genes and other mutational events suffered by duplicated GRBs in teleost genomes permits target gene identification and HCNE/target gene assignment. These findings explain the absence of evolutionary breakpoints from large vertebrate chromosomal segments and will aid in the recognition of position effect mutations within human GRBs.

Se alle arbeider i NVA

• Komisarczuk, Anna Zofia (2026). Genome editing with designer nucleases. Fulltekst i vitenarkiv
• Karabulut, Hatice; Ervik, Carolina Wieczorek; Flister, Renate Sæther; Kopcil, Pavel; Szymanska, Monika & Hedhili, Dalila [Vis alle 12 forfattere av denne artikkelen] (2026). Chasing Better Models: Human HSPC Engraftment in Mouse Models (PRIMA). Fulltekst i vitenarkiv
• Rio, Oline Eriksen; Labun, Kornel; Dahal-Koirala, Shiva; Komisarczuk, Anna Zofia; Valen, Eivind & Haapaniemi, Emma Maria (2026). SNIPSNP: precision design of CRISPR reagents. Fulltekst i vitenarkiv
• Karabulut, Hatice; Ervik, Carolina Wieczorek; Flister, Renate Sæther; Kopcil, Pavel; Szymanska, Monika & Hedhili, Dalila [Vis alle 12 forfattere av denne artikkelen] (2026). Chasing Better Models: Human HSPC Engraftment in Mouse Models. Fulltekst i vitenarkiv
• Ervik, Carolina Wieczorek; Kopcil, Pavel; Szymanska, Monika; Kristiansen, Oddrun; Mamia, Katariina Aino Inkeri & Dahal-Koirala, Shiva [Vis alle 11 forfattere av denne artikkelen] (2025). ENHANCING CRISPR GENE CORRECTION IN HEMATOPOIETIC STEM/PROGENITOR CELLS. Fulltekst i vitenarkiv Vis sammendrag

  CRISPR/Cas9 is a powerful tool for precise gene correction of monogenic blood diseases, such as deficiency of adenosine deaminase 2 (DADA2), a rare disorder that causes recurrent inflammation and vascular damage. However, the efficiency of homology-directed repair (HDR) in CD34+ hematopoietic stem/progenitor cells (HSPCs) is low and limits the therapeutic potential of this approach. In this study, we used a Finnish founder mutation (R169Q) in ADA2 as a model locus for gene correction. We introduced four silent single nucleotide polymorphisms (SNPs) by electroporation using RNPs (ribonucleoproteins) and single-stranded oligodeoxynucleotide (ssODN) repair template in wild-type CD34+ cells isolated from umbilical cord blood. We evaluated the effect of IDT HDR Enhancer v2, and two PKc inhibitors (KU0060684 and AZD7648) on enhancing HDR in CD34+ cells. We measured the colony forming unit (CFU) output of edited cells was compared: a solid assay based on morphology and pigment of cells, and a liquid assay based on cell surface markers (CD14, CD15, CD235), enabling a higher-throughput setup. Using droplet digital PCR as a method to quantify edited cells, we found that PKc inhibitors increased HDR up to 80% in CD34+ cells. Our results suggest that PKc inhibitors is a promising HDR modulator for gene correction in HSPCs. Viability and proliferation were affected in IDT and KU0060684, but in AZD7648 function as well as mock and control. In conclusion, our preliminary results indicates that AZD7648 could lead to the development of more effective gene therapies for monogenic blood diseases in the future. Furthermore, we are optimizing the in vitro culturing of the long-term repopulating hematopoietic stem cells to keep stemness and potentially expanding this population. While initial in vivo testing has been performed, we will also create a novel human xenograft model for modelling diseases e.g. monogenetic disease and leukemia that are irradiation free.

• Ervik, Carolina Wieczorek; Kopcil, Pavel; Szymanska, Monika; Mamia, Katariina Aino Inkeri; Dahal-Koirala, Shiva & Komisarczuk, Anna Zofia [Vis alle 10 forfattere av denne artikkelen] (2025). Enhancing CRISPR gene correction in hematopoetic stem/progenitor cells. doi: https:/iuis2025.org/. Fulltekst i vitenarkiv Vis sammendrag

  CRISPR/Cas9 is a powerful tool for precise gene correction of monogenic blood diseases, such as deficiency of adenosine deaminase 2 (DADA2), a rare disorder that causes recurrent inflammation and vascular damage. However, the efficiency of homology-directed repair (HDR) in CD34+ hematopoietic stem/progenitor cells (HSPCs) is low and limits the therapeutic potential of this approach. In this study, we used a Finnish founder mutation (R169Q) in ADA2 as a model locus for gene correction. We introduced four silent single nucleotide polymorphisms (SNPs) by electroporation using RNPs (ribonucleoproteins) and single-stranded oligodeoxynucleotide (ssODN) repair template in wild-type CD34+ cells isolated from umbilical cord blood. We evaluated the effect of IDT HDR Enhancer v2, and two PKc inhibitors (KU0060684 and AZD7648) on enhancing HDR in CD34+ cells. We measured the colony forming unit (CFU) output of edited cells was compared: a solid assay based on morphology and pigment of cells, and a liquid assay based on cell surface markers (CD14, CD15, CD235), enabling a higher-throughput setup. Using droplet digital PCR as a method to quantify edited cells, we found that PKc inhibitors increased HDR up to 80% in CD34+ cells. Our results suggest that PKc inhibitors is a promising HDR modulator for gene correction in HSPCs. Viability and proliferation were affected in IDT and KU0060684, but in AZD7648 function as well as mock and control. In conclusion, our preliminary results indicates that AZD7648 could lead to the development of more effective gene therapies for monogenic blood diseases in the future. Furthermore, we are optimizing the in vitro culturing of the long-term repopulating hematopoietic stem cells to keep stemness and potentially expanding this population. While initial in vivo testing has been performed, we will also create a novel human xenograft model for modelling diseases e.g. monogenetic disease and leukemia that are irradiation free.

• Haugen, Frida Loe; Mamia, Katariina Aino Inkeri; Rio, Oline Eriksen; Sollano, Anniken Solveig Matheson; Li, Zhuokun & Labun, Kornel [Vis alle 20 forfattere av denne artikkelen] (2025). CRISPR/Cas9-based pipeline to introduce custom edits in autologous T cells. Fulltekst i vitenarkiv
• Haugen, Frida Loe; Mamia, Katariina Aino Inkeri; Rio, Oline Eriksen; Sollano, Anniken Solveig Matheson; Li, Zhuokun & Labun, Kornel [Vis alle 20 forfattere av denne artikkelen] (2025). CRISPR-Cas9 pipeline for custom editing in autologous T cells. doi: https:/event.fourwaves.com/crisprmed25/abstracts/b5911e8e-de8e-4ee4-b8e2-9f08cc2f31d8. Fulltekst i vitenarkiv Vis sammendrag

  CRISPR/Cas9 gene editing technology is a promising tool for correcting pathogenic variants in Inborn Errors of Immunity (IEIs). Current IEI correction strategies rely on cDNA knock-in or gene knockout, which can disrupt gene expression and need extensive optimization. To address this, we developed a safe, efficient CRISPR/Cas9-based T cell editing pipeline using HDR for SNV correction. We observe a rapid and selective expansion of CD3+ T cells from patient peripheral blood following an 8-day cytokine stimulation. To enhance HDR outcomes, we explored multiple strategies and developed an optimized T cell pipeline capable of achieving up to 80% HDR. We found no pre-malignant off-target effects or karyotypic, transcriptomic, or proteomic abnormalities in patient T cells analysed with GUIDE-seq, scRNA-seq, long-read WGS, and proteomics. We observed functional correction of the disease phenotype in patient T cells demonstrated on transcriptomic level by single cell RNA sequencing, on proteomic level by Mass Spectrometry and in cell culture-based assays. So far, we have implemented the pipeline and corrected 12 unique SNV mutations causing 8 diverse IEIs. This platform is designed to streamline CRISPR tool discovery/optimization and can be further developed into a GMP-compatible, scaled-up process for salvage therapy in T cell-related IEIs.

• Kopcil, Pavel; Ervik, Carolina Wieczorek; Mamia, Katariina Aino Inkeri; Dahal-Koirala, Shiva; Szymanska, Monika & Komisarczuk, Anna Zofia [Vis alle 8 forfattere av denne artikkelen] (2024). Enhancing CRISPR Gene Correction In Hematopoietic Stem/Progenitor Cells. Fulltekst i vitenarkiv Vis sammendrag

  CRISPR/Cas9 is a powerful tool for precise gene correction of monogenic blood diseases, such as deficiency of adenosine deaminase 2 (DADA2), a rare disorder that causes recurrent inflammation and vascular damage. However, the efficiency of homology-directed repair (HDR) in CD34+ hematopoietic stem/progenitor cells (HSPCs) is low and limits the therapeutic potential of this approach. In this study, we used a Finnish founder mutation (R169Q) in ADA2 as a model locus for gene correction. We introduced four silent single nucleotide polymorphisms (SNPs) by electroporation using RNPs (ribonucleoproteins) and single-stranded oligodeoxynucleotide (ssODN) repair template in wild-type CD34+ cells isolated from umbilical cord blood. We evaluated the effect of Compound X, a small molecule inhibitor selected from a chemical compounds screen in T cells, on enhancing HDR in CD34+ cells. We also compared two methods for measuring the colony forming unit (CFU) output of edited cells: a solid assay based on morphology and pigment of cells, and a liquid assay based on cell surface markers (CD14, CD15, CD235), enabling a higher-throughput setup. Using droplet digital PCR (ddPCR) as a method to quantify edited cells, we found that Compound X increased HDR up to 80% in CD34+ cells, but reduced CFU output by 2-3 folds. Our results suggest that Compound X is a promising HDR modulator for gene correction in HSPCs, and could lead to the development of more effective gene therapies for monogenic blood diseases in the near future; however, its side effects need to be further investigated. We are currently performing in vivo experiments with Compound X and exploring novel editing strategies, while also working to align our protocols with current good manufacturing practice (cGMP) standards. Furthermore, we aim to assess and optimize the efficacy of Compound X, as well as similar compounds, in long-term repopulating hematopoietic stem cells (LT-HSCs), given their significant clinical importance and the crucial need to enhance editing efficiency in these cells.

• Kopcil, Pavel; Ervik, Carolina Wieczorek; Mamia, Katariina Aino Inkeri; Dahal-Koirala, Shiva; Szymanska, Monika & Komisarczuk, Anna Zofia [Vis alle 8 forfattere av denne artikkelen] (2024). Enhancing CRISPR Gene Correction in Hematopoietic Stem/Progenitor Cells. Fulltekst i vitenarkiv Vis sammendrag

  CRISPR/Cas9 is a powerful tool for precise gene correction of monogenic blood diseases, such as deficiency of adenosine deaminase 2 (DADA2), a rare disorder that causes recurrent inflammation and vascular damage. However, the efficiency of homology-directed repair (HDR) in CD34+ hematopoietic stem/progenitor cells (HSPCs) is low and limits the therapeutic potential of this approach. In this study, we used a Finnish founder mutation (R169Q) in ADA2 as a model locus for gene correction. We introduced four silent single nucleotide polymorphisms (SNPs) by electroporation using RNPs (ribonucleoproteins) and single-stranded oligodeoxynucleotide (ssODN) repair template in wild-type CD34+ cells isolated from umbilical cord blood. We evaluated the effect of Compound X, a small molecule inhibitor selected from a chemical compounds screen in T cells, on enhancing HDR in CD34+ cells. We also compared two methods for measuring the colony forming unit (CFU) output of edited cells: a solid assay based on morphology and pigment of cells, and a liquid assay based on cell surface markers (CD14, CD15, CD235), enabling a higher-throughput setup. Using droplet digital PCR (ddPCR) as a method to quantify edited cells, we found that Compound X increased HDR up to 80% in CD34+ cells, but reduced CFU output by 2-3 folds. Our results suggest that Compound X is a promising HDR modulator for gene correction in HSPCs, and could lead to the development of more effective gene therapies for monogenic blood diseases in the near future; however, its side effects need to be further investigated. We are currently performing in vivo experiments with Compound X and exploring novel editing strategies, while also working to align our protocols with current good manufacturing practice (cGMP) standards. Furthermore, we aim to assess and optimize the efficacy of Compound X, as well as similar compounds, in long-term repopulating hematopoietic stem cells (LT-HSCs), given their significant clinical importance and the crucial need to enhance editing efficiency in these cells.

• Mamia, Katariina Aino Inkeri; Kolbeinsdottir, Solrun; Li, Zhuokun; Reint, Ganna; Komisarczuk, Anna Zofia & Haugen, Frida Høsøien [Vis alle 21 forfattere av denne artikkelen] (2024). CRISPR/Cas9 T cell editing pipeline for monogenic primary immunodeficiency disorders. Fulltekst i vitenarkiv
• Ervik, Carolina Wieczorek; Kopcil, Pavel; Mamia, Katariina Aino Inkeri; Dahal-Koirala, Shiva; Komisarczuk, Anna Zofia & Bak, Rasmus O. [Vis alle 7 forfattere av denne artikkelen] (2024). Enhancing CRISPR Gene Correction In Hematopoietic Stem/Progenitor Cells. Fulltekst i vitenarkiv Vis sammendrag

  CRISPR/Cas9 is a powerful tool for precise gene correction of monogenic blood diseases, such as deficiency of adenosine deaminase 2 (DADA2), a rare disorder that causes recurrent inflammation and vascular damage. However, the efficiency of homology-directed repair (HDR) in CD34+ hematopoietic stem/progenitor cells (HSPCs) is low and limits the therapeutic potential of this approach. In this study, we used a Finnish founder mutation (R169Q) in ADA2 as a model locus for gene correction. We introduced four silent single nucleotide polymorphisms (SNPs) by electroporation using RNPs (ribonucleoproteins) and single-stranded oligodeoxynucleotide (ssODN) repair template in wild-type CD34+ cells isolated from umbilical cord blood. We evaluated the effect of Compound X, a small molecule inhibitor selected from a chemical compounds screen in T cells, on enhancing HDR in CD34+ cells. We also compared two methods for measuring the colony forming unit (CFU) output of edited cells: a solid assay based on morphology and pigment of cells, and a liquid assay based on cell surface markers (CD14, CD15, CD235), enabling a higher-throughput setup. Using droplet digital PCR (ddPCR) as a method to quantify edited cells, we found that Compound X increased HDR up to 80% in CD34+ cells, but reduced CFU output by 2-3 folds. Our results suggest that Compound X is a promising HDR modulator for gene correction in HSPCs, and could lead to the development of more effective gene therapies for monogenic blood diseases in the near future; however, its side effects need to be further investigated. We are currently performing in vivo experiments with Compound X and exploring novel editing strategies, while also working to align our protocols with current good manufacturing practice (cGMP) standards. Furthermore, we aim to assess and optimize the efficacy of Compound X, as well as similar compounds, in long-term repopulating hematopoietic stem cells (LT-HSCs), given their significant clinical importance and the crucial need to enhance editing efficiency in these cells.

• Mamia, Katariina Aino Inkeri; Kolbeinsdottir, Solrun; Li, Zhuokun; Reint, Ganna; Komisarczuk, Anna Zofia & Haugen, Frida Høsøien [Vis alle 21 forfattere av denne artikkelen] (2024). CRISPR/Cas9 T cell editing pipeline for monogenic primary immunodeficiency disorders. Fulltekst i vitenarkiv
• Haapaniemi, Emma Maria; Dahal-Koirala, Shiva; Komisarczuk, Anna Zofia; Mamia, Katariina Aino Inkeri; Rio, Oline Eriksen & Haugen, Frida Høsøien [Vis alle 10 forfattere av denne artikkelen] (2024). Presentation of precision pediatrics and gene editing group. Fulltekst i vitenarkiv
• Mamia, Katariina Aino Inkeri; Kolbeinsdottir, Solrun; Li, Zhuokun; Reint, Ganna; Komisarczuk, Anna Zofia & Haugen, Frida Høsøien [Vis alle 21 forfattere av denne artikkelen] (2024). CRISPR/Cas9 T cell editing pipeline for monogenic primary immunodeficiency disorders. Fulltekst i vitenarkiv
• Komisarczuk, Anna Zofia (2023). Genome editing with designer nucleases. Fulltekst i vitenarkiv
• Komisarczuk, Anna Zofia (2023). CRISPR technology in advanced medicine. Fulltekst i vitenarkiv
• Rio, Oline Eriksen; Komisarczuk, Anna Zofia; Labun, Kornel & Haapaniemi, Emma Maria (2023). CRISPR-Cas9 corrected T cells for personalized primary immunodeficiency disease (PIDD) therapy. Fulltekst i vitenarkiv
• Rio, Oline Eriksen; Komisarczuk, Anna Zofia; Labun, Kornel & Haapaniemi, Emma Maria (2023). CRISPR-Cas9 corrected T cells for personalized primary immunodeficiency disease (PIDD) therapy. Fulltekst i vitenarkiv
• Rio, Oline Eriksen; Haapaniemi, Emma Maria; Komisarczuk, Anna Zofia & Labun, Kornel (2023). CRISPR-Cas9 corrected T cells for personalized primary immunodeficiency disease (PIDD) therapy. Fulltekst i vitenarkiv
• Ervik, Carolina Wieczorek; Kopcil, Pavel; Mamia, Katariina Aino Inkeri; Dahal-Koirala, Shiva; Komisarczuk, Anna Zofia & Bak, Rasmus O. [Vis alle 7 forfattere av denne artikkelen] (2023). Enhancing CRISPR Gene Correction In Hematopoietic Stem/Progenitor Cells. Fulltekst i vitenarkiv
• Ervik, Carolina Wieczorek; Kopcil, Pavel; Mamia, Katariina Aino Inkeri; Dahal-Koirala, Shiva; Komisarczuk, Anna Zofia & Bak, Rasmus O. [Vis alle 7 forfattere av denne artikkelen] (2023). Enhancing CRISPR Gene Correction In Hematopoietic Stem/Progenitor Cells. Fulltekst i vitenarkiv
• Kopcil, Pavel; Ervik, Carolina Wieczorek; Mamia, Katariina Aino Inkeri; Dahal-Koirala, Shiva; Komisarczuk, Anna Zofia & Bak, Rasmus O. [Vis alle 7 forfattere av denne artikkelen] (2023). Enhancing CRISPR gene correction in hematopoietic stem/progenitor cells. Fulltekst i vitenarkiv
• Kopcil, Pavel; Ervik, Carolina Wieczorek; Mamia, Katariina Aino Inkeri; Dahal-Koirala, Shiva; Komisarczuk, Anna Zofia & Bak, Rasmus O [Vis alle 7 forfattere av denne artikkelen] (2023). Enhancing CRISPR Gene Correction In Hematopoietic Stem/Progenitor Cells. Fulltekst i vitenarkiv
• Komisarczuk, Anna Zofia; Kongshaug, Heidi; Li, Ming & Nilsen, Frank (2020). Author Correction: RNAi mediated myosuppressin deficiency affects muscle development and survival in the salmon louse (Lepeophtheirus salmonis) (Scientific Reports, (2019), 9, 1, (6944), 10.1038/s41598-019-43515-w). Scientific Reports. 10(1). doi: 10.1038/s41598-020-69891-2. Fulltekst i vitenarkiv
• Komisarczuk, Anna Zofia (2018). OSMO_LUS - Osmoregulation in Salmon Lice. Fulltekst i vitenarkiv
• Komisarczuk, Anna Zofia (2017). Osmoregulation in Salmon Louse (OSMO-LUS). Fulltekst i vitenarkiv
• Komisarczuk, Anna Zofia (2016). Identification and characterization of three myosuppressin receptors from the parasitic copepod Lepeophtheirus salmonis. Fulltekst i vitenarkiv
• Komisarczuk, Anna Zofia (2015). Her har de klart å lure lakselusen til å velge feil fisk. [Internet]. SYSLA. Fulltekst i vitenarkiv
• Komisarczuk, Anna Zofia (2015). How are salmon lice able to identify the host? Fulltekst i vitenarkiv
• Komisarczuk, Anna Zofia; Grotmol, Sindre & Nilsen, Frank (2013). Unique Organisation of Chemosensory System in Salmon Louse (L.Salmonis) Indicates Adaptation To Strictly Parasitic Life Style. Fulltekst i vitenarkiv
• Zhao, Xiaofeng; Kikuta, Hiroshi; Komisarczuk, Anna Zofia; Becker, Thomas S. & Fjose, Anders (2007). Novel enhancer trap lines identify zebrafish genes associated with development of neural and sensory tissues. Fulltekst i vitenarkiv
• Borg, Nora Helene Hammersten; Komisarczuk, Anna Zofia; Falnes, Pål & Haapaniemi, Emma Maria (2026). Development of a Multimodal Screening Platform for Comparative Evaluation of Prime Editing Efficiency. Establishing an experimental framework through reporter cell line generation, prime editing vector construction, and detection assay development. Universitetet i Oslo. Fulltekst i vitenarkiv Vis sammendrag

  --- Abstract will be published: 2027-05-19

• Komisarczuk, Anna Zofia; Ganeshan, Janaarthan; Haapaniemi, Emma Maria & Winge, Per (2024). epegRNA Screening Platform for Primary Immunodeficiency Mutations in HEK293 cells and Hematopoietic Stem and Progenitory Cells (HSPCs). Norges teknisk-naturvitenskapelige universitet. Fulltekst i vitenarkiv Vis sammendrag

  Mangel på adenosindeaminase (DADA2) er en primær immunsvikt forårsaket av autosomale recessive mutasjoner i adenosindeaminasegen (CECR1, ADA2). Korrekte behandlingsmetoder er først og fremst avhengig av sykdomsfenotypen, og understreker behovet for målrettet genkorreksjon. Prime editing (PE) er et allsidig genomredigeringsverktøy som er i stand til å introdusere innsetting, sletting, alle 12 typer nukleotidendringer og kombinasjoner derav. PE kan potensielt korrigere opptil 90 % av genetiske lidelser, inkludert primære immunsvikt. Evaluering av redigeringseffektiviteten til prime-redigeringsguidRNA (pegRNA) krever imidlertid betydelig innsats. I denne studien beskriver vi en optimalisert prime-redigeringsscreeningsplattform for å vurdere redigeringseffektiviteten til grunnleggende (pegRNA) og konstruert pegRNA (epegRNA) rettet mot pR169Q-mutasjon i ADA2 (ENSG00000093072). Vi evaluerte sekvensriktigheten til epegRNA- og pegRNA-plasmidbiblioteket ved Sanger-sekvensering og genererte lentivirusbiblioteker. Vi evaluerte også den smittsomme titeren ved bruk av digital dråpe-PCR (ddPCR), genererte cellebiblioteker for stabil ekspresjon av epegRNA og pegRNA, og optimaliserte PCR-forholdene for dyp amplikonsekvensering. Vi demonstrerte en ddPCR-basert tilnærming for å kvantifisere lentivirus-kopinummer med høy sensitivitet og lavt standardavvik. Det dynamiske området for ddPCR var mellom 300 og 30 000 målkopier per reaksjon, som ble brukt til estimering av kopiantallvariasjon, beregning av infeksiøs titer og infeksjonsmangfold for epegRNA- og pegRNA-cellebibliotek-preparat. Vi observerte at PCR-tilstanden brukt med epegRNA-plasmid ikke var forenlig med gDNA oppnådd fra cellebiblioteket når biblioteket ble klargjort for amplikonsekvensering. Oppsummert la denne studien grunnlaget for pålitelig og effektiv screening av epegRNA og pegRNA. Vi fremhevet viktigheten av å optimalisere PCR-forhold for amplikonsekvensering. Implementering av denne optimaliserte plattformen lover å effektivisere prosessen med å teste pegRNA-er for monogene mutasjoner.

• Boccara, Charlotte; Saigal, Ruchi; Korchynska, Solomiia & Komisarczuk, Anna Zofia (2023). Optimizing cellular tools to engineer a CRISPR-mediated REM-sleep deprivation model. OsloMet. Fulltekst i vitenarkiv
• Komisarczuk, Anna Zofia (2008). cis–regulation and mis–regulation – Insights into genomic regulatory mechanisms underlying the control of fgf8a in zebrafish Danio rerio. Universitetet i Bergen. ISSN 9788230806098. Fulltekst i vitenarkiv Vis sammendrag

  Fibroblast growth factor 8 (Fgf8) is a potent vertebrate morphogen that plays decisive roles in multiple developmental processes, such as cellular proliferation, survival, differentiation, growth and migration. Fgf8 regionalizes the forebrain and has organizing activity in the midbrain–hindbrain boundary (MHB). Fgf8 induces limb formation and maintains the apical ectodermal ridge (AER), it controls development of the branchial arches and craniofacial skeleton, and its expression in the presomitic mesoderm (PSM) regulates axis elongation, and defines somite boundaries. It follws that the spatiotemporal activity and expression levels of Fgf8 should be tightly controlled, and this control likely occurs at multiple levels – from transcription to regulation of signaling pathways triggered by Fgf8. This thesis constitutes an analysis of mechanisms controlling the activity of Fgf8, and shows that slight modifications in its expression can alter multiple phenotypic traits. Comparative analysis of mammal:teleost conserved chromosomal regions combined with enhancer detection revealed functional chromosomal units called genomic regulatory blocks (GRBs). GRBs usually contain a developmental control gene, or target gene, whose expression is under the control of numerous highly conserved noncoding elements (HCNEs) distributed over a large area that can stretch into and beyond adjacent phylogenetically and functionally unrelated genes, called bystander genes, expressed in patterns unrelated to the target gene. Rearrangements in evolutionarily conserved GRB may have serious consequences during development and thereby contribute to human developmental disease. In all vertebrates, as well as the ascidian Ciona intestinalis, the Fgf8 gene is located in a block of conserved synteny, which also contains the bystander gene Fbxw4. The teleost genomes contain an additional gene upstream of fgf8, slc2A5, which was lost from the mammalian lineage, while the fish have lost BTRC, which is found in all tetrapod genomes. In addition, a whole genome duplication in the teleost lineage resulted in two fgf8 paralogs, fgf8a and –b. Along the fgf8a synteny block a number of HCNEs was detected, located in an area of about 200kb, including inside and around adjacent genes. Functional analysis of these HCNEs in transgenic zebrafish lines suggested that all of them acting as enhancers directed expression of the reporter GFP protein into domains characteristic for fgf8a expression, but not for fbxw4 or slc2a5. Even though in teleosts the fgf8a locus is inverted with respect to tetrapods, and HCNEs are located on the other site of the gene as a result, they still exhibit specific fgf8a activity. Therefore, fgf8a and the bystander genes fbxw4 and slc2a5 genes and cis–regulatory elements of fgf8a constitute a genomic regulatory block (GRB), conserved in all teleost genomes. Insertion of tumor viruses in the proximity of developmental control genes can cause misregulation of the gene and lead to cancer in the mouse. Long terminal repeats (LTRs) of murine tumor viruses contain steroid response elements, which can act as a long–range enhancers. Analysis of four enhancer detection lines in the zebrafish, where integrations of engineered murine proviral vectors had occurred in a 100kb region around the fgf8a gene, caused multiple developmental defects, which were linked to LTR activity. While the expression pattern of YFP reporter protein in all fgf8aCLGY lines mimicked, at least partially, the endogenous expression domains characteristic of fgf8a, fish from the transgenic lines showed extensive phenotypic disorders, including pigmentation anomalies, craniofacial, axial and dermal skeleton defects, and abnormal accumulation of subcutaneous and visceral fat. These phenotypic changes were connected to elevated expression of fgf8a and became manifest during metamorphosis. In addition, transgenic fish had an 80% higher risk of malignant lesions in the brain. The strength of the observed phenotype appeared to be dependent on the distance of the integration site from fgf8a. Progression of tumorigenesis tended to be more severe in males of one line, suggesting androgens as mediators of altered fgf8a activity. Morpholino knock–down experiments targeted to androgen receptor transcript in this transgenic line could reduce the observed elevated fgf8a level. Fgf8 signaling is also regulated at the protein level by specific antagonists, the sprouty proteins. A new member of this family was identified in zebrafish by enhancer detection. A transgenic line was isolated with an expression pattern overlapping fgf8a in forebrain, dorsal diencephalon, MHB, branchial arches, pectoral fin and PSM. Mapping by inverse PCR identified this locus as sprouty1. Sprouty1 is a downstream modulator of Fgf signaling and its expression is attenuated by the small molecular inhibitor of FGF receptor 1, SU5402. Together these findings highlight complex regulatory mechanisms underlying the control, in all vertebrates, of Fgf8, a potent morphogen, which controls multiple aspects of animal form and function.

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