Biology subjects

Crysnanto, D.

Publications and source records attributed to Crysnanto, D..

12 recordsLinked to original sources

bioRxiv2025

Artemis Regulates Homology-Independent Prime Editing (PRINS) for Enhanced Genomic Insertions

Nuclease-based prime editing (PEn) offers enhanced genomic insertion efficiency compared to nickase-based prime editors, but its reliance on double-strand break (DSB) repair leads to complex and often unpredictable on-target indel distributions. PRINS editing, a PEn variant utilizing springRNAs, uniquely relies on non-homologous end joining (NHEJ) for insertions, providing an insertion-only strategy ideal for functional protein tagging or serine integrase landing pad insertions, yet it suffers from inherent imprecision. Here, we identify the DNA repair factor Artemis (DCLRE1C) as a key regulator of PEn-generated indel profiles, particularly in springRNA-mediated PRINS editing. Through a targeted genetic screen, we show that the absence of Artemis significantly shifts indel distributions away from deletions and shorter truncations towards longer, functionally acceptable insertions. Our data indicates that Artemis cleaves PEn-generated 3-overhangs in a length-dependent manner, with its impact increasing for longer reverse-transcribed overhangs. This understanding reveals that regulating Artemis activity can improve insertion frequency specifically for PRINS. We develop and validate robust epigenetic (CRISPRoff) and antisense oligonucleotide (ASO) strategies to effectively silence/knockdown Artemis expression, successfully recapitulating the beneficial PRINS editing outcomes observed in Artemis-deficient cells. Leveraging these insights, we show that Artemis modulation can enhance endogenous protein tagging in cells, including challenging hiPSC-derived non-dividing cardiomyocytes. Our findings support Artemis as a key regulator of PRINS editing outcomes and present a tunable strategy to optimize insertion efficiency for diverse genomic engineering and therapeutic applications.

molecular biology↗

bioRxiv2022

Graph construction method impacts variation representation and analyses in a bovine super-pangenome

Several models and algorithms have been proposed to build pangenomes from multiple input assemblies, but their impact on variant representation, and consequently downstream analyses, is largely unknown. We create multi-species "super-pangenomes" using pggb, cactus, and minigraph with the Bos taurus taurus reference sequence and eleven haplotype-resolved assemblies from taurine and indicine cattle, bison, yak, and gaur. We recover 221k nonredundant structural variations (SVs) from the pangenomes, of which 135k (61%) are common to all three. SVs derived from assembly-based calling show high agreement with the consensus calls from the pangenomes (96%), but validate only a small proportion of variations private to each graph. Pggb and cactus, which also incorporate base-level variation, have approximately 95% exact matches with assembly-derived small variant calls, which significantly improves the edit rate when realigning assemblies compared to minigraph. We use the three pangenomes to investigate 9,566 variable number tandem repeats (VNTRs), finding 63% have identical predicted repeat counts in the three graphs, while minigraph can over or underestimate the count given its approximate coordinate system. We examine a highly variable VNTR locus and show that repeat unit copy number impacts expression of proximal genes and non-coding RNA. Our findings indicate good consensus between the three pangenome methods but also show their individual strengths and weaknesses that need to be considered when analysing different types of variants from multiple input assemblies.

genomics↗

bioRxiv2021

Bovine pangenome reveals trait-associated structural variation from diverse assembly inputs

Advantages of pangenomes over linear reference assemblies for genome research have recently been established. However, potential effects of sequence platform and assembly approach, or of combining assemblies created by different approaches, on pangenome construction have not been investigated. We generated haplotype-resolved assemblies from the offspring of three bovine trios representing increasing levels of heterozygosity that each demonstrate a substantial improvement in contiguity, completeness, and accuracy over the current Bos taurus reference genome. Diploid coverage as low as 20x for HiFi or 60x for ONT was sufficient to produce two haplotype-resolved assemblies meeting the standards set by the Vertebrate Genome Project. Structural variant-based pangenomes created from the haplotype-resolved assemblies demonstrated significant consensus regardless of sequence platform, assembler algorithm, or coverage. Inspecting pangenome topologies identified 90 thousand structural variants including 931 overlapping with coding sequences; this approach revealed variants affecting QRICH2, PRDM9, HSPA1A, TAS2R46, and GC that have potential to affect phenotype.

genomics↗

bioRxiv2021

Characterization of a haplotype-reference panel for genotyping by low-pass sequencing in Swiss Large White pigs

BackgroundThe key-ancestor approach has been frequently applied to prioritize individuals for whole-genome sequencing based on their marginal genetic contribution to current populations. Using this approach, we selected 70 key ancestors from two lines of the Swiss Large White breed that have been selected divergently for fertility and fattening traits and sequenced their genomes with short paired-end reads. ResultsUsing pedigree records, we estimated the effective population size of the dam and sire line to 72 and 44, respectively. In order to assess sequence variation in both lines, we sequenced the genomes of 70 boars at an average coverage of 16.69-fold. The boars explained 87.95 and 95.35% of the genetic diversity of the breeding populations of the dam and sire line, respectively. Reference-guided variant discovery using the GATK revealed 26,862,369 polymorphic sites. Principal component, admixture and FST analyses indicated considerable genetic differentiation between the lines. Genomic inbreeding quantified using runs of homozygosity was higher in the sire than dam line (0.28 vs 0.26). Using two complementary approaches (CLR and iHS), we detected 51 signatures of selection. However, only six signatures of selection overlapped between both lines. We used the sequenced haplotypes of the 70 key ancestors as a reference panel to call 22,618,811 genotypes in 175 pigs that had been sequenced at very low coverage (1.11-fold) using GLIMPSE. The genotype concordance, non-reference sensitivity and non-reference discrepancy between thus inferred and Illumina PorcineSNP60 BeadChip-called genotypes was 97.60, 98.73 and 3.24%, respectively. The low-pass sequencing-derived genomic relationship coefficients were highly correlated (r > 0.99) with those obtained from microarray genotyping. ConclusionsWe assessed genetic diversity within and between two lines of the Swiss Large White pig breed. Our analyses revealed considerable differentiation, even though the split into two populations occurred only few generations ago. The sequenced haplotypes of the key ancestor animals enabled us to implement genotyping by low-pass sequencing which offers an intriguing cost-effective approach to increase the variant density over current array-based genotyping by more than 350-fold.

genomics↗

bioRxiv2021

Novel functional sequences uncovered through a bovine multi-assembly graph

Linear reference genomes are typically assembled from single individuals. They are unable to reflect the genetic diversity of populations and lack millions of bases. To overcome such limitations and make non-reference sequences amenable to genetic investigations, we build a multi-assembly graph from six reference-quality assemblies from taurine cattle and their close relatives. We uncover 70,329,827 bases that are missing in the bovine linear reference genome. The missing sequences encode novel transcripts that are differentially expressed between individual animals. Reads which were previously poorly or unmapped against the bovine reference genome now align accurately to the non-reference sequences. We show that the non-reference sequences contain polymorphic sites that segregate within and between breeds of cattle. Our efforts to uncover novel functional sequences from a multi-assembly graph pave the way towards the transition to a more representative bovine reference genome.

genomics↗

bioRxiv2020

Deletion of porcine BOLL causes defective acrosomes and subfertility in Yorkshire boars

A recessively inherited sperm defect of Finnish Yorkshire boars was detected more than a decade ago. Affected boars produce ejaculates that contain many spermatozoa with defective acrosomes resulting in low fertility and small litters. The acrosome defect was mapped to porcine chromosome 15 but the causal mutation has not been identified. We re-analyzed microarray-derived genotypes of affected boars and performed a haplotype-based association study. Our results confirmed that the acrosome defect maps to a 12.24 Mb segment of porcine chromosome 15 (P=3.38 x 10-14). In order to detect the mutation causing defective acrosomes, we sequenced the genomes of two affected and three unaffected boars to an average coverage of 11-fold. Read-depth analysis revealed a 55 kb deletion that segregates with the acrosome defect. The deletion encompasses the BOLL gene encoding the boule homolog, RNA binding protein which is an evolutionarily highly conserved member of the DAZ (deleted in azoospermia) gene family. Lack of BOLL expression causes spermatogenic arrest and sperm maturation failure in many species. Our study reveals that absence of BOLL is associated with a sperm defect also in pigs. The acrosomes of boars that carry the deletion in the homozygous state are defective suggesting that lack of porcine BOLL compromises acrosome formation. Our findings warrant further research to investigate the precise function of BOLL during spermatogenesis and sperm maturation in pigs.

genomics↗

bioRxiv2020

A 63-bp insertion in exon 2 of the porcine KIF21A gene is associated with arthrogryposis multiplex congenita

Arthrogryposis multiplex congenita (AMC) is a recessively inherited fatal disease detected almost 20 years ago in the Swiss Large White pig population. A diagnostic marker test enabled the identification of carrier animals, but the underlying causal mutation remains unknown. To identify the mutation underlying AMC, we collected whole-genome genotyping and sequencing data for 11 affected piglets and 23 healthy pigs. Haplotype-based case-control association testing using 47,829 SNPs confirmed that AMC maps to SSC5 (P = 9.4 x10-13). Subsequent autozygosity mapping revealed a common 6.06 Mb region (from 66,757,970 to 72,815,151 bp) of extended homozygosity in 11 piglets affected by AMC. We detected a 63-bp insertion in the second exon of KIF21A gene encoding Kinesin Family Member 21A using whole-genome sequences of a carrier boar, two of its affected and two heterozygous piglets. This insertion was compatible with the recessive inheritance of AMC. The 63-bp insertion likely represents a loss-of-function allele because it is predicted to introduce a premature stop codon in KIF21A gene (p.Val41_Phe42insTer) that truncates 1,614 amino acids ([~] 97%) from the protein. Lack of KIF21A protein is lethal in mice, thus providing additional evidence that a loss-of function allele of KIF21A might cause fatal AMC in pigs. We found that this deleterious allele still segregates at low frequency in the Swiss Large White pig population. The unambiguous detection of carrier animals can now facilitate the eradication of the deleterious allele from the population.

genomics↗

bioRxiv2020

Activation of cryptic splicing in bovine WDR19 is associated with reduced semen quality and male fertility

Cattle are ideally suited to investigate the genetics of male reproduction, because semen quality and fertility are recorded for all ejaculates of artificial insemination bulls. We analysed 26,090 ejaculates of 794 Brown Swiss bulls to assess ejaculate volume, sperm concentration, sperm motility, sperm head and tail anomalies and insemination success. The heritability of the six semen traits was between 0 and 0.26. Genome-wide association testing on 607,511 SNPs revealed a QTL on bovine chromosome 6 that was associated with sperm motility (P = 2.5 x 10-27), head (P = 2.0 x 10-44) and tail anomalies (P = 7.2 x 10-49) and insemination success (P = 9.9 x 10-13). The QTL harbors a recessive allele that compromises semen quality and male fertility. We replicated the effect of the QTL on fertility (P = 7.1 x 10-32) in an independent cohort of 2481 Brown Swiss bulls. The analysis of whole-genome sequencing data revealed that a synonymous variant (BTA6:58373887C>T, rs474302732) in WDR19 encoding WD repeat-containing protein 19 was in linkage disequilibrium with the fertility-associated haplotype. WD repeat-containing protein 19 is a constituent of the intraflagellar transport complex that is essential for the physiological function of motile cilia and flagella. Bioinformatic and transcription analyses revealed that the BTA6:58373887 T-allele activates a cryptic exonic splice site that eliminates three evolutionarily conserved amino acids from WDR19. Western blot analysis demonstrated that the BTA6:58373887 T-allele decreases protein expression. We make the remarkable observation that, in spite of negative effects on semen quality and bull fertility, the BTA6:58373887 T-allele has a frequency of 24 % in the Brown Swiss population. Our findings are the first to uncover a variant that is associated with quantitative variation in semen quality and male fertility in cattle. Author summaryIn cattle farming, artificial insemination is the most commonly used method of breeding. To ensure high fertilization rates, ejaculate quality and insemination success are closely monitored in artificial insemination bulls. We analyse semen quality, insemination success and microarray-called genotypes at more than 600,000 genome-wide SNP markers in 794 bulls to identify a recessive allele that compromises semen quality. We take advantage of whole-genome sequencing to pinpoint a variant in the coding sequence of WDR19 encoding WD repeat-containing protein 19 that activates a novel exonic splice site. Our results indicate that cryptic splicing in WDR19 is associated with reduced male reproductive performance. This is the first report of a variant that contributes to quantitative variation in bovine semen quality.

genomics↗

bioRxiv2019

Sequence read mapping and variant discovery from bovine breed-specific augmented reference graphs

BackgroundThe current bovine genomic reference sequence was assembled from the DNA of a Hereford cow. The resulting linear assembly lacks diversity because it does not contain allelic variation. Lack of diversity is a drawback of linear references that causes reference allele bias. High nucleotide diversity and the separation of individuals by hundreds of breeds make cattle ideally suited to investigate the optimal composition of variation-aware references. ResultsWe augment the bovine linear reference sequence (ARS-UCD1.2) with variants filtered for allele frequency in dairy (Brown Swiss, Holstein) and dual-purpose (Fleckvieh, Original Braunvieh) cattle breeds to construct either breed-specific or pan-genome reference graphs using the vg toolkit. We find that read mapping is more accurate to variation-aware than linear references if pre-selected variants are used to construct the genome graphs. Graphs that contain random variants do not improve read mapping over the linear reference sequence. Breed-specific augmented and pan-genome graphs enable almost similar mapping accuracy improvements over the linear reference. We construct a whole-genome graph that contains the Hereford-based reference sequence and 14 million alleles that have alternate allele frequency greater than 0.03 in the Brown Swiss cattle breed. We show that our novel variation-aware reference facilitates accurate read mapping and unbiased sequence variant genotyping for SNPs and Indels. ConclusionsWe developed the first variation-aware reference graph for an agricultural animal: https://doi.org/10.5281/zenodo.3759712. Our novel reference structure improves sequence read mapping and variant genotyping over the linear reference. Our work is a first step towards the transition from linear to variation-aware reference structures in species with high genetic diversity and many sub-populations.

genomics↗

bioRxiv2019

Assessing genomic diversity and signatures of selection in Original Braunvieh cattle using whole-genome sequencing data

BackgroundAutochthonous cattle breeds represent an important source of genetic variation because they might carry alleles that enable them to adapt to local environment and food conditions. Original Braunvieh (OB) is a local cattle breed of Switzerland used for beef and milk production in alpine areas. Using whole-genome sequencing (WGS) data of 49 key ancestors, we characterize genomic diversity, genomic inbreeding, and signatures of selection in Swiss OB cattle at nucleotide resolution. ResultsWe annotated 15,722,811 million SNPs and 1,580,878 million Indels including 10,738 and 2,763 missense deleterious and high impact variants, respectively, that were discovered in 49 OB key ancestors. Six Mendelian trait-associated variants that were previously detected in breeds other than OB, segregated in the sequenced key ancestors including variants causal for recessive xanthinuria and albinism. The average nucleotide diversity (1.6 x 10-3) was higher in OB than many mainstream European cattle breeds. Accordingly, the average genomic inbreeding quantified using runs of homozygosity (ROH) was relatively low (FROH=0.14) in the 49 OB key ancestor animals. However, genomic inbreeding was higher in more recent generations of OB cattle (FROH=0.16) due to a higher number of long (> 1 Mb) runs of homozygosity. Using two complementary approaches, composite likelihood ratio test and integrated haplotype score, we identified 95 and 162 genomic regions encompassing 136 and 157 protein-coding genes, respectively, that showed evidence (P < 0.005) of past and ongoing selection. These selection signals were enriched for quantitative trait loci related to beef traits including meat quality, feed efficiency and body weight and pathways related to blood coagulation, nervous and sensory stimulus. ConclusionsWe provide a comprehensive overview of sequence variation in Swiss OB cattle genomes. With WGS data, we observe higher genomic diversity and less inbreeding in OB than many European mainstream cattle breeds. Footprints of selection were detected in genomic regions that are possibly relevant for meat quality and adaptation to local environmental conditions. Considering that the population size is low and genomic inbreeding increased in the past generations, the implementation and adoption of optimal mating strategies seems warranted to maintain genetic diversity in the Swiss OB cattle population.

genomics↗

bioRxiv2018

Accurate sequence variant genotyping in cattle using variation-aware genome graphs

BackgroundThe genotyping of sequence variants typically involves as a first step the alignment of sequencing reads to a linear reference genome. Because a linear reference genome represents only a small fraction of sequence variation within a species, reference allele bias may occur at highly polymorphic or diverged regions of the genome. Graph-based methods facilitate to compare sequencing reads to a variation-aware genome graph that incorporates non-redundant DNA sequences that segregate within a species. We compared sensitivity and accuracy of graph-based sequence variant genotyping using the Graphtyper software to two widely used methods, i.e., GATK and SAMtools, that rely on linear reference genomes using whole-genomes sequencing data of 49 Original Braunvieh cattle.\n\nResultsWe discovered 21,140,196, 20,262,913 and 20,668,459 polymorphic sites using GATK, Graphtyper, and SAMtools, respectively. Comparisons between sequence variant and microarray-derived genotypes showed that Graphtyper outperformed both GATK and SAMtools in terms of genotype concordance, non-reference sensitivity, and non-reference discrepancy. The sequence variant genotypes that were obtained using Graphtyper had the lowest number of mendelian inconsistencies for both SNPs and indels in nine sire-son pairs with sequence data. Genotype phasing and imputation using the Beagle software improved the quality of the sequence variant genotypes for all tools evaluated particularly for animals that have been sequenced at low coverage. Following imputation, the concordance between sequence- and microarray-derived genotypes was almost identical for the three methods evaluated, i.e., 99.39, 99.29, and 99.52 % for GATK, SAMtools, and Graphtyper, respectively. Graphtyper required considerably more computing resources than SAMtools but it required less than GATK.\n\nConclusionsSequence variant genotyping using Graphtyper is accurate, sensitive and computationally feasible in cattle. Graph-based methods enable sequence variant genotyping from variation-aware reference genomes that may incorporate cohort-specific sequence variants which is not possible with the current implementations of state-of-the-art methods that rely on linear reference genomes.

genomics↗

bioRxiv2018

Widespread gene duplication and adaptive evolution in the RNA interference pathways of the Drosophila obscura group

BackgroundRNA interference (RNAi) related pathways provide defense against viruses and transposable elements, and have been implicated in the suppression of meiotic drive elements. Genes in these pathways often exhibit high levels of adaptive substitution, and over longer timescales show frequent gene duplication and loss-- most likely as a consequence of their role in mediating conflict with these parasites. This is particularly striking for Argonaute 2 (Ago2), which is ancestrally the key effector of antiviral RNAi in insects, but has repeatedly formed new testis-specific duplicates in the recent history of the Drosophila obscura group.\n\nResultsHere we take advantage of publicly available genomic and transcriptomic data to identify six further RNAi-pathway genes that have duplicated in this clade of Drosophila, and examine their evolutionary history. As seen for Ago2, we observe high levels of adaptive amino-acid substitution and changes in sex-biased expression in many of the paralogs. However, our phylogenetic analysis suggests that co-duplications of the RNAi machinery were not synchronous, and our expression analysis fails to identify consistent male-specific expression.\n\nConclusionsThese results confirm that RNAi genes, including genes of the antiviral and piRNA pathways, undergo frequent independent duplications and that their history has been particularly labile within the Drosophila obscura group. However, they also suggest that the selective pressures driving these changes have not been consistent, implying that more than one selective agent may be responsible.

evolutionary biology↗