Results 1-25 of about 1,000
  1. Adolescent idiopathic scoliosis (AIS) is a common and progressive spinal deformity in children that exhibits striking sexual dimorphism, with girls at more than five-fold greater risk of severe disease compared to boys. Despite its medical impact, the molecular mechanisms that drive AIS are largely unknown. We previously defined a female-specific AIS genetic risk locus in an enhancer near the PAX1 gene. Here we sought to define the roles of PAX1 and newly-identified AIS-associated genes in the developmental mechanism of AIS. In a genetic study of 9,161 individuals with AIS and 80,731 unaffected controls, significant association was identified with a variant in COL11A1 encoding collagen (alpha1) XI (rs3753841; NM_080629_c.4004C>T; p.(Pro1335Leu); P=7.07e-11, OR=1.118). Using CRISPR mutagenesis we generated Pax1 knockout mice (Pax1-/-). In postnatal spines we found that Pax1 and collagen (alpha1) XI protein both localize within the intervertebral disc (IVD)-vertebral junction region encompassing the growth plate, with less collagen (alpha1) XI detected in Pax1-/- spines compared to wildtype. By genetic targeting we found that wildtype Col11a1 expression in growth plate cells (GPCs) suppresses expression of Pax1 and of Mmp3, encoding the matrix metalloproteinase 3 enzyme implicated in matrix remodeling. However, this suppression was abrogated in the presence of the AIS-associated COL11A1P1335L mutant. Further, we found that either knockdown of the estrogen receptor gene Esr2, or tamoxifen treatment, significantly altered Col11a1 and Mmp3 expression in GPCs. These studies support a new molecular model of AIS pathogenesis wherein genetic variation and estrogen signaling increase disease susceptibility by altering a Pax1-Col11a1-Mmp3 signaling axis in the growth plate.
    Date: 2023-05-27
    Authors: Wise C, Yu H, Khanshour AM, Ushiki A, Otomo N, Koike Y, Einarsdottir E, Fan Y, Antunes L, Kidane YH, Sheng R, Zhang Y, Pei J, Grishin NV, Evers BMV, Cheung JPY, Herring JA, Terao C, Song Y, Gurnett C, Gerdhem P, Ikegawa S, Rios JJ, Ahituv N.
    Ref: bioRxiv
  2. Neurodevelopmental disorders have been associated with genetic mutations that affect cellular function, including chromatin regulation and epigenetic modifications. Recent studies in humans have identified mutations in KMT2C, an enzyme responsible for modifying histone tails and depositing H3K4me1 and H3K4me3, as being associated with Kleefstra syndrome 2 and autism spectrum disorder (ASD). However, the precise role of KMT2C mutations in brain disorders remains poorly understood. Here we employed CRISPR/Cas9 gene editing to analyze the effects of KMT2C knockout on animal behavior. Knocking out KMT2C expression in cortical neurons and the mouse brain resulted in decreased KMT2C levels. Importantly, KMT2C knockout animals exhibited repetitive behaviors, social deficits, and intellectual disability resembling ASD. Our findings shed light on the involvement of KMT2C in neurodevelopmental processes and establish a valuable model for elucidating the cellular and molecular mechanisms underlying KMT2C mutations and their relationship to Kleefstra syndrome 2 and ASD.
    Date: 2023-05-26
    Authors: Brauer B, Merino-Veliz N, Ahumada-Marchant C, Arriagada G, Bustos FJ.
    Ref: bioRxiv
  3. Bacteria and archaea acquire resistance to viruses and plasmids by integrating fragments of foreign DNA into the first repeat of a CRISPR array. However, the mechanism of site-specific integration remains poorly understood. Here, we determine a 560 kDa integration complex structure that explains how Cas (Cas1-2/3) and non-Cas proteins (IHF) fold 150 base-pairs of host DNA into a U-shaped bend and a loop that protrude from Cas1-2/3 at right angles. The U-shaped bend traps foreign DNA on one face of the Cas1-2/3 integrase, while the loop places the first CRISPR repeat in the Cas1 active site. Both Cas3s rotate 100-degrees to expose DNA binding sites on either side of the Cas2 homodimer, that each bind an inverted repeat motif in the leader. Leader sequence motifs direct Cas1-2/3-mediated integration to diverse repeat sequences that have a 5'-GT.
    Date: 2023-05-26
    Authors: Santiago-Frangos A, Henriques WS, Wiegand T, Gauvin CC, Buyukyoruk M, Graham AB, Wilkinson RA, Triem L, Neselu K, Eng ET, Lander GC, Wiedenheft B.
    Ref: bioRxiv
  4. Precision gene editing in primary hematopoietic stem and progenitor cells (HSPCs) would facilitate both curative treatments for monogenic disorders as well as disease modelling. Precise efficiencies even with the CRISPR/Cas system, however, remain limited. Through an optimization of guide RNA delivery, donor design, and additives, we have now obtained mean precise editing efficiencies >90% on primary cord blood HSCPs with minimal toxicity. Critically, editing is even across the progenitor hierarchy, and does not substantially distort the hierarchy or affect lineage outputs in colony-forming cell assays. As modelling of many diseases requires heterozygosity, we also demonstrated that the overall editing and zygosity can be tuned by adding in defined mixtures of mutant and wild-type donor. With these optimizations, editing at near-perfect efficiency can now be accomplished directly in human HSPCs. This will open new avenues in both therapeutic strategies and disease modelling.
    Date: 2023-05-26
    Authors: Cloarec-Ung F, Beaulieu J, Suthananthan A, Lehnertz B, Sauvageau G, Sheppard HM, Knapp DJ.
    Ref: bioRxiv
  5. The short-lived African killifish Nothobranchius furzeri lives in seasonal freshwater ponds and has evolved remarkable traits to survive in this limited environment. One of those traits is a genetic XX/XY sex-determination system, which ensures an equal distribution of both sexes. Comparisons of female and male genomic sequences identified the Y-chromosomal copy of the TGF-β family member gdf6 as the candidate male sex-determining (SD) gene, which was named gdf6Y in contrast to the X-chromosomal allele gdf6X. CRISPR/Cas9-mediated inactivation of gdf6Y in N. furzeri led to a complete male-to-female sex reversal in XY animals. The homozygous inactivation of gdf6X on the other hand led to a detrimental phenotype post-hatching. This phenotype was compensated by gdf6Y, revealing that the latter became the SD gene while retaining at least some of its original gdf6 function. Gdf6Y is expressed in testicular somatic cells already prior to hatching, where it represses the germ cell-intrinsic feminizing gene foxl2l. We have identified components of the TGF-β signaling pathway, especially the inhibitor of DNA binding genes id1/2/3, and the mRNA decay activator zfp36l2, as Gdf6Y targets. We conclude that Gdf6Y exerts its function as the male sex-determining gene by suppressing female-specific genes in the developing gonad of male N. furzeri.
    Date: 2023-05-26
    Authors: Richter A, Mörl H, Thielemann M, Kleemann M, Geißen R, Schwarz R, Albertz C, Koch P, Petzold A, Groth M, Hartmann N, Herpin A, Englert C.
    Ref: bioRxiv
  6. Objective: Comparing to the coding sequences (CDS), the 3’-untranslated region (3’-UTR) of PD-L1 is extremely longer but its role and regulators are less explored. Methods: The whole 3’-UTR region was deleted by CRISPR-Cas9. Prognostic analysis was performed using online tools. Immune infiltration analysis was performed using Timer and Xcell package. Immunotherapy response prediction and cox regression were performed using R software. MicroRNA network analysis was conducted by Cytoscape software. Results: The level of PD-L1 was dramatically and significantly up-regulated in 3’-UTR deficient cells. Furthermore, we found a panel of 43 RNA binding proteins (RBPs) that correlated with PD-L1 in a majority of cancer cell lines and tumor tissues. Among them, PARP14 is widely associated with immune checkpoints, tumor microenvironment and immune infiltrating cells in various cancer types. We also identified 38 MicroRNA that associated with PD-L1 across cancers. The miR-3139, miR-4761 and miR-15a-5p are significantly associated with PD-L1 in most of cancer types. Finally, we revealed 21 m6A regulators that have a strong correlation with PD-L1. More importantly, by combing the identified RBPs and m6A regulators, we established a predictive immune signature including RBMS1, QKI, YTHDC1, ZC3HAV1, RBM38 and PPARGC1B to predict the responsiveness of cancer patients upon receiving immune checkpoint blockade. Conclusions: We demonstrated the critical role of 3’-UTR in the regulation of PD-L1 and uncovered a large number of potential PD-L1 regulators in pan-cancer. The generated biomarker signature has power to predict patient’s prognosis, but along with the potential PD-L1 regulators should be further biologically investigated.
    Date: 2023-05-26
    Authors: Chen Z, Pi H, Wang Z, Zhang Y, Zhang J, Xu J, Qu X, Liu L, Shen H, Lu Y, Chen M, Zhang W, Mao R, Sun R, Fan Y.
    Ref: Research Square
  7. N4-acetylcytidine (ac4C) is an epitranscriptomic modification of mRNA that is catalyzed by N-acetyltransferase 10 (NAT10), a critical factor known to influence mRNA stability. However, its role in development has not been investigated. In this study, we used CRISPR/Cas9 and RNAi technology to knock out and knock down nat10 , the zebrafish ortholog of human NAT10, and evaluated their effects on development, behavior, and transcriptome. Our findings indicate that nat10 deficiency in zebrafish embryos results in increased embryo mortality and developmental abnormalities. Additionally, behavioral and histological evaluations revealed that nat10 knockdown led to increased anxiety-like behavior and severe vision impairment. Transcriptome profiling and RT-PCR results showed that nat10 knockdown significantly downregulated the expression of retinal transcripts that are enriched in response to light stimuli, photoreceptors, and visual perception. Furthermore, dot-blot and RIP-PCR analyses confirmed a significant reduction in ac4C levels in total RNA and opsin mRNA in nat10 knockdown zebrafish. Our results highlight the essential role of ac4C in embryonic development, especially in visual development. This zebrafish model could be helpful for studying ac4C modification in neurodevelopmental disorders.
    Date: 2023-05-26
    Authors: Yang H, Zhuo D, Huang Z, Luo G, Liang S, Fan Y, Zhao Y, Lv X, Qiu C, Zhang L, Liu Y, Sun T, Chen X, Li S, Jin X.
    Ref: Research Square
  8. Milk fat globule EGF factor 8 (MFGE8) also known as Lactadherin is a glycoprotein which plays a crucial role in mammary gland remodeling. Our group has previously identified MFGE8 as a marker associated with high milk yielding cows. Here, we have generated MFGE8 knock-out buffalo mammary epithelial cells (BuMEC) via CRISPR-cas9 technology to decipher its role in lactation biology. Among three gRNAs used to generate knock-outs, gRNA3 reduced MFGE8 expression with better efficiency which was confirmed at transcriptomic and proteomic level and the stable knock-out cells obtained were named mfge8-/-/gRNA3. The amplicon sequencing of the edited region using next generation sequencing (NGS) showed that 54% of total reads showed indels, 3-4 bp upstream to PAM site in 2nd exon. To comprehend the role of MFGE8, mfge8-/-/gRNA3 cells were examined for proteome level changes in comparison to wild type cells using an iTRAQ experiment. A total 4282 proteins were identified in mfge8-/-/gRNA3 cells and among them 178 were found to be differentially expressed above and below a threshold of ≥1.5 and ≤0.6. Majority of DEPs were found to be associated with regulation of hydrolase activity, endopeptidase activity and cytoskeletal organization and some DEPs including FABP3, FABP4, FABP5, KNG1, MT2A, CD82 and SERPINH1 belonged to genes associated with milk synthesis. To the best of our knowledge, this is the first study which provides a comprehensive proteome profile of MFGE8 knockout BuMEC and explores the downstream effects of disruption of MFGE8 gene. Overall, the present study will provide new insights into lactation biology.
    Date: 2023-05-26
    Authors: VATS P, Kumar R, Kumar R, Kaushik JK, Mohanty AK, Kumar S.
    Ref: Research Square
  9. CCCTC-binding factor (CTCF), an insulator protein with 11 zinc fingers, is enriched at the boundaries of topologically associated domains (TADs) in eukaryotic genomes. In this study, we isolated and analyzed the cDNAs encoding HpCTCF, the CTCF homolog in the sea urchin Hemicentrotus pulcherrimus, to investigate its expression patterns and functions during early development of sea urchin. HpCTCF contains nine zinc fingers corresponding to fingers 2-10 of the vertebrate CTCF. The expression pattern analysis revealed that HpCTCF mRNA was detected at all developmental stages and in the entire embryo. Upon expressing the HpCTCF-GFP fusion protein in early embryos, we observed its uniform distribution within interphase nuclei. However, during mitosis, it disappeared from the chromosomes and subsequently reassembled on the chromosome during telophase. Moreover, the morpholino-mediated knockdown of HpCTCF resulted in mitotic arrest during the morula-to-blastula stage. Most of the arrested chromosomes were not phospholylated at serine 10 of histone H3, indicating that mitosis was arrested at the telophase by HpCTCF depletion. Furthermore, impaired sister chromatid segregation was observed using time-lapse imaging of HpCTCF-knockdown embryos. Thus, HpCTCF is essential for mitotic progression during the early development of sea urchins, especially during the telophase-to-interphase transition. However, the normal development of pluteus larvae in CRISPR-mediated HpCTCF knockout embryos suggests that disruption of zygotic HpCTCF expression has little effect on embryonic and larval development.
    Date: 2023-05-26
    Authors: Watanabe K, Fujita M, Okamoto K, Yoshioka H, Moriwaki M, Tagashira H, Yamamoto T, Sakamoto N.
    Ref: bioRxiv
  10. Background: Neisseria gonorrhoeae is one of the most common bacterial sexually transmitted infections. The emergence of antimicrobial-resistant N. gonorrhoeae is an urgent public health threat. Currently, diagnosis of N. gonorrhoeae infection requires expensive laboratory infrastructure, while antimicrobial susceptibility determination requires bacterial culture, both of which are infeasible in low-resource areas where prevalence is highest. Recent advances in molecular diagnostics, such as Specific High-sensitivity Enzymatic Reporter unLOCKing (SHERLOCK) using CRISPR-Cas13a and isothermal amplification, have the potential to provide low-cost detection of pathogen and antimicrobial resistance. Methods and Results We designed and optimized RNA guides and primer-sets for SHERLOCK assays capable of detecting N. gonorrhoeae via the porA gene and of predicting ciprofloxacin susceptibility via a single mutation in the gyrase A (gyrA) gene. We evaluated their performance using both synthetic DNA and purified N. gonorrhoeae isolates. For porA, we created both a fluorescence-based assay and lateral flow assay using a biotinylated FAM reporter. Both methods demonstrated sensitive detection of 14 N. gonorrhoeae isolates and no cross-reactivity with 3 non-gonococcal Neisseria isolates. For gyrA, we created a fluorescence-based assay that correctly distinguished between 20 purified N. gonorrhoeae isolates with phenotypic ciprofloxacin resistance and 3 with phenotypic susceptibility. We confirmed the gyrA genotype predictions from the fluorescence-based assay with DNA sequencing, which showed 100% concordance for the isolates studied. Conclusion We report the development of Cas13a-based SHERLOCK assays that detect N. gonorrhoeae and differentiate ciprofloxacin-resistant isolates from ciprofloxacin-susceptible isolates.
    Date: 2023-05-26
    Authors: Allan-Blitz L, Shah P, Adams G, Branda J, Klausner JD, Goldstein R, Sabeti PC, Lemieux JE.
    Ref: medRxiv
  11. Neurodevelopmental disorders are frequently linked to mutations in synaptic organizing molecules. MAM domain containing glycosylphosphatidylinositol anchor 1 and 2 (MDGA1 and MDGA2) are a family of synaptic organizers suggested to play an unusual role as synaptic repressors, but studies offer conflicting evidence for their localization. Using epitope-tagged MDGA1 and MDGA2 knock-in mice, we found that native MDGAs are expressed throughout the brain, peaking early in postnatal development. Surprisingly, endogenous MDGA1 was enriched at excitatory, but not inhibitory, synapses. Both shRNA knockdown and CRISPR/Cas9 knockout of MDGA1 resulted in cell-autonomous, specific impairment of AMPA receptor-mediated synaptic transmission, without affecting GABAergic transmission. Conversely, MDGA2 knockdown/knockout selectively depressed NMDA receptor-mediated transmission but enhanced inhibitory transmission. Our results establish that MDGA2 acts as a synaptic repressor, but only at inhibitory synapses, whereas both MDGAs are required for excitatory transmission. This nonoverlapping division of labor between two highly conserved synaptic proteins is unprecedented
    Date: 2023-05-26
    Authors: Bemben MA, Sandoval MA, Le AA, Won S, Chau VN, Lauterborn JC, Incontro S, Li KH, Burlingame AL, Roche KW, Gall CM, Nicoll RA, Diaz-Alonso J.
    Ref: bioRxiv
  12. Nephron progenitor cells (NPCs) self-renew and differentiate into nephrons, the functional units of the kidney. Here we report manipulation of p38 and YAP activity creates a synthetic niche that allows the long-term clonal expansion of primary mouse and human NPCs, and induced NPCs (iNPCs) from human pluripotent stem cells. Cultured iNPCs resemble closely primary human NPCs, generating nephron organoids with abundant distal convoluted tubule cells, which are not observed in published kidney organoids. The synthetic niche reprograms differentiated nephron cells into NPC state, recapitulating the plasticity of developing nephron in vivo. Scalability and ease of genome-editing in the cultured NPCs allow for genome-wide CRISPR screening, identify-ing novel genes associated with kidney development and disease. A rapid, efficient, and scalable organoid model for polycystic kidney disease was derived directly from genome-edited NPCs, and validated in drug screen. These technological platforms have broad applications to kidney development, disease, plasticity, and regeneration.
    Date: 2023-05-25
    Authors: Huang B, Zeng Z, Li H, Li Z, Chen X, Guo J, Zhang CC, Schreiber ME, Vonk A, Xiang T, Patel T, Li Y, Parvez R, Der B, Chen JH, Liu Z, Thornton ME, Grubbs BH, Diao Y, Dou Y, Gnedeva K, Lindstrom NO, Ying Q, Pastor-Soler NM, Fei T, Hallows KR, McMahon AP, Li Z.
    Ref: bioRxiv
  13. A missense variant in the tetratricopeptide repeat domain 3 ( TTC3 ) gene (rs377155188, p.S1038C, NM_003316.4:c.3113C>G) was found to segregate with disease in a multigenerational family with late onset Alzheimer′s disease. This variant was introduced into induced pluripotent stem cells (iPSCs) derived from a cognitively intact individual using CRISPR genome editing and the resulting isogenic pair of iPSC lines were differentiated into cortical neurons. Transcriptome analysis showed an enrichment for genes involved in axon guidance, regulation of actin cytoskeleton, and GABAergic synapse. Functional analysis showed that the TTC3 p.S1038C iPSC-derived neuronal progenitor cells had altered 3D morphology and increased migration, while the corresponding neurons had longer neurites, increased branch points, and altered expression levels of synaptic proteins. Pharmacological treatment with small molecules that target the actin cytoskeleton could revert many of these cellular phenotypes, suggesting a central role for actin in mediating the cellular phenotypes associated with the TTC3 p.S1038C variant.
    Date: 2023-05-25
    Authors: Cukier HN, Duarte CL, Laverde-Paz MJ, Simon SA, Van Booven DJ, Miyares AT, Whitehead PL, Hamilton-Nelson KL, Adams LD, Carney RM, Cuccaro ML, Vance JM, Pericak-Vance MA, Griswold AJ, Dykxhoorn DM.
    Ref: bioRxiv
  14. Studies of the young gene Heterochromatin Protein 6 (HP6) have challenged the dogma that essential functions are only seen in genes with a long evolutionary history. Based on its prominent expression in Drosophila germ cells, we asked if HP6 might play a role in germline development. Surprisingly, we found that CRISPR-generated HP6 null mutants are viable and fertile. We identified an independent lethal allele and an RNAi off-target effect that prevented accurate interpretation of HP6 essentiality in previous studies. We found that the vast majority of young essential genes were viable when tested with orthologous methods. Together our data call into question the frequency with which young genes gain essential functions.
    Date: 2023-05-25
    Authors: Grill S, Riley AM, Selvaraj M, Lehmann R.
    Ref: bioRxiv
  15. Single-cell transcriptomics (scRNA-seq) has revolutionized our understanding of cell types and states in various contexts, such as development and disease. To selectively capture protein-coding polyadenylated transcripts, most methodologies rely on poly(A) enrichment to exclude ribosomal transcripts that constitute >80% of the transcriptome. However, it is common for ribosomal transcripts to sneak into the library, which can add significant background by flooding libraries with irrelevant sequences. The challenge of amplifying all RNA transcripts from a single cell has motivated the development of new technologies to optimize retrieval of transcripts of interest. This problem is especially striking in planarians, where a single 16S ribosomal transcript is widely enriched (20-80%) across single-cell methods. Therefore, we adapted the Depletion of Abundant Sequences by Hybridization (DASH) to the standard 10X scRNA-seq protocol. We designed single-guide RNAs tiling the 16S sequence for CRISPR-mediated degradation, and subsequently generated untreated and DASH-treated datasets from the same libraries to enable a side-by-side comparison of the effects of DASH. DASH specifically removes 16S sequences without off-target effects on other genes. By assessing the cell barcodes shared by both libraries, we find that DASH-treated cells have consistently higher complexity given the same amount of reads, which enables the detection of a rare cell cluster and more differentially expressed genes. In conclusion, DASH can be easily integrated into existing sequencing protocols and customized to deplete unwanted transcripts in any organism.
    Date: 2023-05-25
    Authors: Wang K, Adler CE.
    Ref: bioRxiv
  16. SYNGAP1 is a Ras-GTPase activating protein highly enriched at excitatory synapses in the brain. De novo loss-of-function mutations in SYNGAP1 are a major cause of genetically defined neurodevelopmental disorders (NDD). These mutations are highly penetrant and cause SYNGAP1-related intellectual disability (SRID), an NDD characterized by cognitive impairment, social deficits, early-onset seizures, and sleep disturbances. Studies in rodent neurons have shown that Syngap1 regulates developing excitatory synapse structure and function, and heterozygous Syngap1 knockout mice have deficits in synaptic plasticity, learning and memory, and have seizures. However, how specific SYNGAP1 mutations found in humans lead to disease has not been investigated in vivo. To explore this, we utilized CRISPR-Cas9 system to generate knock-in mouse models with two distinct known causal variants of SRID including a frameshift mutation leading to a premature stop codon, SYNGAP1; L813RfsX22, and a single- nucleotide mutation in an intron that creates a cryptic splice acceptor site leading to premature stop codon, SYNGAP1; c.3583-9G>A. While reduction in Syngap1 mRNA varies from 30-50% depending on the specific mutation, both models show ~50% reduction in Syngap1 protein, have deficits in synaptic plasticity, and recapitulate key features of SRID including hyperactivity and impaired working memory. These data suggest that half the amount of SYNGAP1 protein is key to the pathogenesis of SRID. These results provide a resource and establish a framework for the development of therapeutic strategies for this disorder.
    Date: 2023-05-25
    Authors: Araki Y, Gerber EE, Rajkovich KE, Hong I, Johnson RC, Lee H, Kirkwood A, Huganir RL.
    Ref: bioRxiv
  17. Summary Despite recent advances in the treatment of melanoma, many patients with metastatic disease still succumb to their disease. To identify tumor-intrinsic modulators of immunity to melanoma, we performed a whole-genome CRISPR screen in melanoma and identified multiple components of the HUSH complex, including Setdb1 , as hits. We found that loss of Setdb1 leads to increased immunogenicity and complete tumor clearance in a CD8+ T-cell dependent manner. Mechanistically, loss of Setdb1 causes de-repression of endogenous retroviruses (ERVs) in melanoma cells and triggers tumor-cell intrinsic type-I interferon signaling, upregulation of MHC-I expression, and increased CD8+ T-cell infiltration. Furthermore, spontaneous immune clearance observed in Setdb1 -/- tumors results in subsequent protection from other ERV-expressing tumor lines, supporting the functional anti-tumor role of ERV-specific CD8+ T-cells found in the Setdb1 -/- microenvironment. Blocking the type-I interferon receptor in mice grafted with Setdb1 -/- tumors decreases immunogenicity by decreasing MHC-I expression, leading to decreased T-cell infiltration and increased melanoma growth comparable to Setdb1 wt tumors. Together, these results indicate a critical role for Setdb1 and type-I interferons in generating an inflamed tumor microenvironment, and potentiating tumor-cell intrinsic immunogenicity in melanoma. This study further emphasizes regulators of ERV expression and type-I interferon expression as potential therapeutic targets for augmenting anti-cancer immune responses.
    Date: 2023-05-24
    Authors: McGeary MK, Damsky W, Daniels A, Song E, Micevic G, Huet-Calderwood C, Lou HJ, Paradkar S, Kaech S, Calderwood DA, Turk BE, Iwasaki A, Bosenberg MW.
    Ref: bioRxiv
  18. Chromosomal instability and copy number alterations (CNAs) are pervasive in human cancers. However, the exact timing and mechanism of CNA formation during carcinogenesis remain poorly understood. Here, we describe scCUTseq, an agile and robust workflow for spatially resolved single-cell CNA profiling which could be applied to characterize the CNA landscape in tumor samples. Importantly, scCUTseq could clearly resolve the copy number profiles of different cell lines and was free of cross-contamination, highlighting its specificity. Secondly, we assessed the sensitivity of scCUTseq by determining its ability to detect a 7Mb deletion induced by CRISPR-Cas9 in a small percentage (~3%) of cells transfected with two small guide RNAs (sgRNA) targeting the KMT2A and HYLS1 locus on chr11. In 3.3% of the cells transfected with both sgRNAs, scCUTseq detected a single copy of the exact 7 Mb deletion, in line with the quantification by FISH, highlighting the sensitivity of our method. Lastly, to rule out the artifacts introduced by the MALBAC step, we compared scCUTseq with Acoustic Cell Tagmentation (ACT), another single-cell SCNA profiling method that is based on DNA tagmentation and does not involve a whole genome amplification (WGA) step. Both visual and quantitative comparisons revealed that the genome-wide copy number profiles obtained by either method were highly similar, indicating that the WGA step in scCUTseq does not result in obvious artifact CNA calls. scCUTseq is a versatile and scalable method, which can greatly facilitate the depiction of the single-cell CNA profile in cancers.
    Date: 2023-05-24
    Authors: Zhang N, Simonetti M, Harbers L, Helleday T, Roukos V, Marchio C, Bienko M, Crosetto N.
    Ref: Research Square
  19. CRISPR screens with single-cell transcriptomic readouts are a valuable tool to understand the effect of genetic perturbations, but are currently limited because genotypes are inferred from the guide RNA identity. We have developed a technique that couples single-cell genotyping to transcriptomics of the same cells to enable screening for the effects of single nucleotide variants. Analysis of variants tiling across the JAK1 gene demonstrates the importance of determining the precise genetic perturbation and classifies missense variants into three functional categories.
    Date: 2023-05-24
    Authors: Cooper SE, Coelho MA, Strauss ME, Gontarczyk AM, Wu Q, Garnett MJ, Marioni JC, Bassett AR.
    Ref: bioRxiv
  20. Fluorescence reporter strains of human malaria parasites are powerful tools to study the interaction of the parasites with both human and mosquito hosts. However, low fluorescence intensity in transmission-relevant parasite stages and the choice of insertion loci that cause parasite developmental defects in the mosquito largely limits usefulness of currently available lines. To overcome these limitations, we used a CRISPR-Cas9-mediated approach to generate PfOBC13 GFP , a novel selection marker-free reporter parasite in the background of the African NF54 Plasmodium falciparum line. As docking site, we selected the OBC13 locus that is dispensable for asexual and sexual development in vitro . PfOBC13 GFP parasites encode GFP flanked by hsp70 UTRs that drive strong fluorescence reporter expression throughout blood and mosquito stages, enabling parasite detection by such high throughput methods as flow cytometry. When compared to the parental line, PfOBC13 GFP parasites showed normal development during blood and mosquito stages, and they efficiently infected the main African vector Anopheles coluzzii, overcoming one of the limitations of the previously developed fluorescent reporter lines based on the Pfs47 locus. PfOBC13 GFP constitutes a potent tool enabling host-pathogen studies throughout Plasmodium life cycle. Importance Fluorescence reporter strains have been very useful in malaria research, however, up to date they had limitations in mosquito infectivity and fluorescence intensity. Here we report the generation of PfOBC13 GFP , a new fluorescent parasite strain of the human malaria parasite P. falciparum . PfOBC13 GFP parasites are highly fluorescent throughout the life cycle, making them an ideal tool for the study the parasite progression through blood and mosquito stages. They efficiently infect the African mosquito vector A. coluzzii , allowing the study of this African parasite in its biological background. Moreover, strong parasite fluorescence enables flow cytometry and live microscopy characterization of all parasite stages, especially those involved in transmission.
    Date: 2023-05-24
    Authors: Suárez-Cortés P, Costa G, Andres M, Eyermann D, Kreschel C, Spohr L, Goosmann C, Brinkmann V, Levashina EA.
    Ref: bioRxiv
  21. The fate of DNA double-strand breaks (DSBs) generated by the Cas9 nuclease has been thoroughly studied. Repair via non-homologous end-joining (NHEJ) or homologous recombination (HR) is the common outcome. However, little is known about unrepaired DSBs and the type of damage they can trigger in plants. In this work, we designed a new assay that detects loss of heterozygosity (LOH) in somatic cells, enabling the study of a broad range of DSB-induced genomic events. The system relies on a mapped phenotypic marker which produces a light purple color (Betalain pigment) in all plant tissues. Plants with sectors lacking the Betalain marker upon DSB induction between the marker and the centromere were tested for LOH events. Using this assay we detected a flower with a twin yellow and dark purple sector, corresponding to a germinally transmitted somatic crossover event. We also identified instances of small deletions of genomic regions spanning the T-DNA and whole chromosome loss. In addition, we show that major chromosomal rearrangements including loss of large fragments, inversions, and translocations were clearly associated with the CRISPR-induced DSB. Detailed characterization of complex rearrangements by whole genome sequencing, molecular, and cytological analyses, supports a model in which breakage-fusion-bridge cycle followed by chromothripsis-like rearrangements had been induced. Our LOH assay provides a new tool for precise breeding via targeted crossover detection. It also uncovers CRISPR mediated chromothripsis-lke events that had not been previously identified in plants.
    Date: 2023-05-24
    Authors: Samach A, Mafessoni F, Gross O, Melamed-Bessudo C, Filler-Hayut S, Dahan-Meir T, Amsellem Z, Pawlowski WP, Levy AA.
    Ref: bioRxiv
  22. Group B Streptococcus (GBS; S. agalactiae ) causes chorioamnionitis, neonatal sepsis, and can also cause disease in healthy or immunocompromised adults. GBS possesses a type II-A CRISPR-Cas9 system, which defends against foreign DNA within the bacterial cell. Several recent publications have shown that GBS Cas9 influences genome-wide transcription through a mechanism uncoupled from its function as a specific, RNA-programmable endonuclease. We examine GBS Cas9 effects on genome-wide transcription through generation of several isogenic variants with specific functional defects. We compare whole-genome RNA-seq from Δ cas9 GBS with a full-length Cas9 gene deletion; dcas9 defective in its ability to cleave DNA but still able to bind to frequently occurring protospacer adjacent motifs; and scas9 that retains its catalytic domains but is unable to bind protospacer adjacent motifs. Comparing scas9 GBS to the other variants, we identify nonspecific protospacer adjacent motif binding as a driver of genome-wide, Cas9 transcriptional effects in GBS. We also show that Cas9 transcriptional effects from nonspecific scanning tend to influence genes involved in bacterial defense and nucleotide or carbohydrate transport and metabolism. While genome-wide transcription effects are detectable by analysis of next-generation sequencing, they do not result in virulence changes in a mouse model of sepsis. We also demonstrate that catalytically inactive dCas9 expressed from the GBS chromosome can be used with a straightforward, plasmid-based, single guide RNA expression system to suppress transcription of specific GBS genes without potentially confounding off-target effects. We anticipate that this system will be useful for study of nonessential and essential gene roles in GBS physiology and pathogenesis.
    Date: 2023-05-24
    Authors: Gopalakrishna KP, Hillebrand GH, Bhavana VH, Elder JL, D’Mello A, Tettelin H, Hooven TA.
    Ref: bioRxiv
  23. ABSTRACT MicroRNAs are small, non-coding RNA molecules that regulate expression of their target genes. The MIR444 gene family is present exclusively in monocotyledons, and microRNAs444 from this family have been shown to target certain MADS-box transcription factors in rice and barley. We identified three barley MIR444 ( MIR444a / b / c ) genes and comprehensively characterized their structure and the processing pattern of the primary transcripts (pri-miRNAs444). Pri-microRNAs444 undergo extensive alternative splicing, by which functional and non-functional pri-miRNA444 isoforms are generated. We show that barley pri-miRNAs444 contain numerous open reading frames (ORFs) whose transcripts associate with ribosomes. Using specific antibodies, we provide evidence that selected ORFs encoding PEP444a within MIR444a and PEP444c within MIR444c are expressed in barley plants. Moreover, we demonstrate that CRISPR-associated endonuclease 9 (Cas9)-mediated mutagenesis of the PEP444c encoding sequence results in a decreased level of PEP444 transcript in barley shoots and roots, and a 5-fold reduced level of mature microRNA444c in roots. Taken together, our observations suggest that PEP444c encoded by the MIR444c gene is involved in microRNA444c biogenesis in barley.
    Date: 2023-05-24
    Authors: Chojnacka A, Smoczynska A, Bielewicz D, Pacak A, Hensel G, Kumlehn J, Karlowski W, Grabsztunowicz M, Sobieszczuk-Nowicka E, Jarmolowski A, Szweykowska-Kulinska Z.
    Ref: bioRxiv
  24. Understanding how single cells divide and differentiate into different cell types in developed organs is one of the major tasks of developmental and stem cell biology. Recently, lineage tracing technology using CRISPR/Cas9 genome editing have enabled simultaneous readouts of gene expressions and lineage barcodes in single cells, which allows for the reconstruction of the cell division tree, and even the detection of cell types and differentiation trajectories at the whole organism level. While most state-of-the-art methods for lineage reconstruction utilize only the lineage barcode data, methods that incorporate gene expression data are emerging, aiming to improve the accuracy of lineage reconstruction. However, effectively incorporating the gene expression data requires a reasonable model on how gene expression data changes along generations of divisions. Here, we present LinRace (Lineage Reconstruction with asymmetric cell division model), a method that integrates the lineage barcode and gene expression data using the asymmetric cell division model and infers cell lineage under a framework combining Neighbor Joining and maximum-likelihood heuristics. On both simulated and real data, LinRace outputs more accurate cell division trees than existing methods for lineage reconstruction. Moreover, LinRace can output the cell states (cell types) of ancestral cells, which is rarely performed with existing lineage reconstruction methods. The information on ancestral cells can be used to analyze how a progenitor cell generates a large population of cells with various functionalities. LinRace is available at: https://github.com/ZhangLabGT/LinRace.
    Date: 2023-05-24
    Authors: Zhang X, Pan X, Li H, Putta P.
    Ref: Research Square
  25. The heterochronic genes of C. elegans comprise the best-studied pathway controlling the timing of tissue and organ formation in an animal. To begin to understand the evolution of this pathway and the significance of the relationships among its components, we characterized 11 C. briggsae orthologs of C. elegans heterochronic genes. Using CRISPR/Cas9, we made a variety of alleles and found that several mutant phenotypes differ in significant ways from those of C. elegans . Although most mutant orthologs displayed defects in developmental timing, their phenotypes could differ in which stages were affected, the penetrance and expressivity of the phenotypes, or by having additional pleiotropies that were not obviously connected to developmental timing. However, when examining pairwise epistasis and synergistic relationships, we found those paralleled the known relationships between their C. elegans orthologs, suggesting that the arrangements of these genes in functional modules is conserved, but the modules’ relationships to each other and/or to their targets has drifted since the time of the species’ last common ancestor. Furthermore, our investigation has revealed a relationship to this pathway to other aspects of the animal’s growth and development, including gonad development, that is relevant to both species.
    Date: 2023-05-23
    Authors: Ivanova M, Moss EG.
    Ref: bioRxiv
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