Efficient CRISPR-Cas9-Mediated Knock-In of Composite Tags in Zebrafish Using Long ssDNA as a Donor

Despite the unprecedented gene editing capability of CRISPR-Cas9-mediated targeted knock-in, the efficiency and precision of this technology still require further optimization, particularly for multicellular model organisms, such as the zebrafish (Danio rerio). Our study demonstrated that an ∼200 base-pair sequence encoding a composite tag can be efficiently “knocked-in” into the zebrafish genome using a combination of the CRISPR-Cas9 ribonucleoprotein complex and a long single-stranded DNA (lssDNA) as a donor template. Here, we targeted the sox3, sox11a, and pax6a genes to evaluate the knock-in efficiency of lssDNA donors with different structures in somatic cells of injected embryos and for their germline transmission. The structures and sequence characteristics of the lssDNA donor templates were found to be crucial to achieve a high rate of precise and heritable knock-ins. The following were our key findings: (1) lssDNA donor strand selection is important; however, strand preference and its dependency appear to vary among the target loci or their sequences. (2) The length of the 3′ homology arm of the lssDNA donor affects knock-in efficiency in a site-specific manner; particularly, a shorter 50-nt arm length leads to a higher knock-in efficiency than a longer 300-nt arm for the sox3 and pax6a knock-ins. (3) Some DNA sequence characteristics of the knock-in donors and the distance between the CRISPR-Cas9 cleavage site and the tag insertion site appear to adversely affect the repair process, resulting in imprecise editing. By implementing the proposed method, we successfully obtained precisely edited sox3, sox11a, and pax6a knock-in alleles that contained a composite tag composed of FLAGx3 (or PAx3), Bio tag, and HiBiT tag (or His tag) with moderate to high germline transmission rates as high as 21%. Furthermore, the knock-in allele-specific quantitative polymerase chain reaction (qPCR) for both the 5′ and 3′ junctions indicated that knock-in allele frequencies were higher at the 3′ side of the lssDNAs, suggesting that the lssDNA-templated knock-in was mediated by unidirectional single-strand template repair (SSTR) in zebrafish embryos.

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GAMIBHEAR: whole-genome haplotype reconstruction from Genome Architecture Mapping data

Bioinformatics ◽

10.1093/bioinformatics/btab238 ◽

2021 ◽

Author(s):

Julia Markowski ◽

Rieke Kempfer ◽

Alexander Kukalev ◽

Ibai Irastorza-Azcarate ◽

Gesa Loof ◽

...

Keyword(s):

Genetic Variants ◽

Embryonic Stem Cell Line ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cell ◽

Supplementary Information ◽

Model Organisms ◽

Genome Architecture ◽

Chromatin Conformation ◽

Haplotype Reconstruction ◽

Allele Specific

Abstract Motivation Genome Architecture Mapping (GAM) was recently introduced as a digestion- and ligation-free method to detect chromatin conformation. Orthogonal to existing approaches based on chromatin conformation capture (3C), GAM’s ability to capture both inter- and intra-chromosomal contacts from low amounts of input data makes it particularly well suited for allele-specific analyses in a clinical setting. Allele-specific analyses are powerful tools to investigate the effects of genetic variants on many cellular phenotypes including chromatin conformation, but require the haplotypes of the individuals under study to be known a-priori. So far however, no algorithm exists for haplotype reconstruction and phasing of genetic variants from GAM data, hindering the allele-specific analysis of chromatin contact points in non-model organisms or individuals with unknown haplotypes. Results We present GAMIBHEAR, a tool for accurate haplotype reconstruction from GAM data. GAMIBHEAR aggregates allelic co-observation frequencies from GAM data and employs a GAM-specific probabilistic model of haplotype capture to optimise phasing accuracy. Using a hybrid mouse embryonic stem cell line with known haplotype structure as a benchmark dataset, we assess correctness and completeness of the reconstructed haplotypes, and demonstrate the power of GAMIBHEAR to infer accurate genome-wide haplotypes from GAM data. Availability GAMIBHEAR is available as an R package under the open source GPL-2 license at https://bitbucket.org/schwarzlab/gamibhear Maintainer [email protected] Supplementary information Supplementary information is available at Bioinformatics online.

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Rapid evolution at the Drosophila telomere: transposable element dynamics at an intrinsically unstable locus

Genetics ◽

10.1093/genetics/iyaa027 ◽

2020 ◽

Vol 217 (2) ◽

Author(s):

Michael P McGurk ◽

Anne-Marie Dion-Côté ◽

Daniel A Barbash

Keyword(s):

Copy Number ◽

Large Scale ◽

Insertion Site ◽

Rapid Evolution ◽

Interspecific Variation ◽

High Rate ◽

Evolutionary Strategy ◽

Control Mechanisms ◽

Genetic Conflict ◽

Number Variation

AbstractDrosophila telomeres have been maintained by three families of active transposable elements (TEs), HeT-A, TAHRE, and TART, collectively referred to as HTTs, for tens of millions of years, which contrasts with an unusually high degree of HTT interspecific variation. While the impacts of conflict and domestication are often invoked to explain HTT variation, the telomeres are unstable structures such that neutral mutational processes and evolutionary tradeoffs may also drive HTT evolution. We leveraged population genomic data to analyze nearly 10,000 HTT insertions in 85 Drosophila melanogaster genomes and compared their variation to other more typical TE families. We observe that occasional large-scale copy number expansions of both HTTs and other TE families occur, highlighting that the HTTs are, like their feral cousins, typically repressed but primed to take over given the opportunity. However, large expansions of HTTs are not caused by the runaway activity of any particular HTT subfamilies or even associated with telomere-specific TE activity, as might be expected if HTTs are in strong genetic conflict with their hosts. Rather than conflict, we instead suggest that distinctive aspects of HTT copy number variation and sequence diversity largely reflect telomere instability, with HTT insertions being lost at much higher rates than other TEs elsewhere in the genome. We extend previous observations that telomere deletions occur at a high rate, and surprisingly discover that more than one-third do not appear to have been healed with an HTT insertion. We also report that some HTT families may be preferentially activated by the erosion of whole telomeres, implying the existence of HTT-specific host control mechanisms. We further suggest that the persistent telomere localization of HTTs may reflect a highly successful evolutionary strategy that trades away a stable insertion site in order to have reduced impact on the host genome. We propose that HTT evolution is driven by multiple processes, with niche specialization and telomere instability being previously underappreciated and likely predominant.

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A red herring in zebrafish genetics: allele-specific gene expression can underlie altered transcript abundance in zebrafish mutants

10.1101/2021.08.06.455380 ◽

2021 ◽

Author(s):

Richard J White ◽

Eirinn Mackay ◽

Stephen W Wilson ◽

Elisabeth M Busch-Nentwich

Keyword(s):

Differentially Expressed Genes ◽

Differentially Expressed Gene ◽

Transcript Abundance ◽

Differentially Expressed ◽

Model Organisms ◽

Specific Gene ◽

Wild Type ◽

Specific Expression ◽

Genomic Location ◽

Allele Specific

In model organisms, RNA sequencing is frequently used to assess the effect of genetic mutations on cellular and developmental processes. Typically, animals heterozygous for a mutation are crossed to produce offspring with different genotypes. Resultant embryos are grouped by genotype to compare homozygous mutant embryos to heterozygous and wild-type siblings. Genes that are differentially expressed between the groups are assumed to reveal insights into the pathways affected by the mutation. Here we show that in zebrafish, differentially expressed genes are often overrepresented on the same chromosome as the mutation due to different levels of expression of alleles from different genetic backgrounds. Using an incross of haplotype-resolved wild-type fish, we found evidence of widespread allele-specific expression, which appears as differential expression when comparing embryos homozygous for a region of the genome to their siblings. When analysing mutant transcriptomes, this means that differentially expressed genes on the same chromosome as a mutation of interest may not be caused by that mutation. Typically, the genomic location of a differentially expressed gene is not considered when interpreting its importance with respect to the phenotype. This could lead to pathways being erroneously implicated or overlooked due to the noise of spurious differentially expressed genes on the same chromosome as the mutation. These observations have implications for the interpretation of RNA-seq experiments involving outbred animals and non-inbred model organisms.

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Pervasive head-to-tail insertions of DNA templates mask desired CRISPR-Cas9–mediated genome editing events

Science Advances ◽

10.1126/sciadv.aax2941 ◽

2020 ◽

Vol 6 (7) ◽

pp. eaax2941 ◽

Cited By ~ 10

Author(s):

Boris V. Skryabin ◽

Delf-Magnus Kummerfeld ◽

Leonid Gubar ◽

Birte Seeger ◽

Helena Kaiser ◽

...

Keyword(s):

Conditional Knockout ◽

Nonhomologous End Joining ◽

High Rate ◽

Model Organisms ◽

Pcr Analysis ◽

End Joining ◽

Dna Templates ◽

Homology Directed Repair ◽

Conditional Knockout Mouse ◽

Wide Range

CRISPR-Cas9–mediated homology-directed DNA repair is the method of choice for precise gene editing in a wide range of model organisms, including mouse and human. Broad use by the biomedical community refined the method, making it more efficient and sequence specific. Nevertheless, the rapidly evolving technique still contains pitfalls. During the generation of six different conditional knockout mouse models, we discovered that frequently (sometimes solely) homology-directed repair and/or nonhomologous end joining mechanisms caused multiple unwanted head-to-tail insertions of donor DNA templates. Disturbingly, conventionally applied PCR analysis, in most cases, failed to identify these multiple integration events, which led to a high rate of falsely claimed precisely edited alleles. We caution that comprehensive analysis of modified alleles is essential and offer practical solutions to correctly identify precisely edited chromosomes.

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Yolk Protein Synthesis in the Ovary of Octopus Vulgaris and its Control by the Optic Gland Gonadotropin

Journal of Experimental Biology ◽

10.1242/jeb.59.3.665 ◽

1973 ◽

Vol 59 (3) ◽

pp. 665-674

Author(s):

R. K. O'DOR ◽

M. J. WELLS

Keyword(s):

Protein Synthesis ◽

High Rate ◽

Follicle Cells ◽

Blood Protein ◽

Octopus Vulgaris ◽

Yolk Proteins ◽

Stage Of Development ◽

Rapid Accumulation ◽

A Site

1. Over 98% of a dose of [14C]leucine injected into the circulation of Octopus vulgaris is removed from the blood during the first hour. 2. There is a rapid accumulation of labelled protein in the ovaries of maturing animals within 2 h of injection. Within 5-7 h the ovaries contain nearly 40% of the injected label in protein form. 3. Removal of the optic glands prevents this accumulation of protein. 4. There is little labelled protein in the livers of either control or maturing animals at any time; but a slow, steady accumulation occurs in their blood. 5. The level of labelled protein appearing in the blood of acutely ovariectomized, maturing females is no higher than in controls; and when blood protein from ovariectomized animals is injected into normal maturing females it is not taken up by the ovaries. 6. The labelled protein which accumulates in the blood is probably haemocyanin. Preliminary experiments indicate that the branchial glands, which are already believed to be a site of haemocyanin synthesis on morphological grounds, show a high rate of protein synthesis and release. 7. Isolated ovarian follicles in a liquid medium synthesize protein at a rate somewhat lower, but comparable with, the apparent in vivo rate. 8. The combined evidence from these experiments indicates that in Octopus yolk proteins are formed within the ovary-probably by the follicle cells-rather than being synthesized elsewhere and transported through the blood, as in arthropods and vertebrates. 9. The optic gland gonadotropin is essential for maintenance of protein synthesis during secondary vitellogenesis and the follicle cells are a likely site for its action during this stage of development.

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Event-specific quantitative polymerase chain reaction methods for detection of double-herbicide-resistant genetically modified corn MON 87419 based on the 3′-junction of the insertion site

Bioscience Biotechnology and Biochemistry ◽

10.1093/bbb/zbab040 ◽

2021 ◽

Author(s):

Likun Long ◽

Wei Yan ◽

Congcong Li ◽

Liming Dong ◽

Na Liu ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Genetically Modified ◽

Quantitative Polymerase Chain Reaction ◽

Insertion Site ◽

Chain Reaction ◽

Herbicide Resistant ◽

Qualitative Polymerase Chain Reaction ◽

Relative Standard ◽

Junction Site ◽

Polymerase Chain

ABSTRACT MON 87419 was one of the new transgenic corn events developed in US with the trait of herbicide resistance to both dicamba and glyphosate. To monitor unintended release of genetically modified organism in the future, as well as to meet GM-labeling requirements, it is requisite to develop a reliable method for the detection and quantification of MON 87419, an event-specific primer pair was designed to amplify the 3′-junction site between the endogenous genome sequence and the transferred DNA of GM event MON 87419, amplicons of desired size were produced by qualitative polymerase chain reaction (PCR) assay. For the validation of this quantitative method, the mixed samples containing 10%, 1%, and 0.1% MON 87419 ingredient were quantified. The precisions were expressed as relative standard deviations, deviated by 7.87%, 12.94%, and 19.98%, respectively. These results clearly demonstrate that the PCR methods we developed herein can be used for event-specific quantitative testing of the double-herbicide-resistant corn MON 87419.

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High-Resolution Genetic Mapping With Ordered Arrays of Saccharomyces cerevisiae Deletion Mutants

Genetics ◽

10.1093/genetics/162.3.1091 ◽

2002 ◽

Vol 162 (3) ◽

pp. 1091-1099 ◽

Cited By ~ 4

Author(s):

Paul Jorgensen ◽

Bryce Nelson ◽

Mark D Robinson ◽

Yiqun Chen ◽

Brenda Andrews ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

High Resolution ◽

Genetic Mapping ◽

Large Scale ◽

Model Organisms ◽

Deletion Mutants ◽

Wild Type ◽

Allele Specific ◽

Single Allele ◽

Kinase Signaling Pathway

Abstract We present a method for high-resolution genetic mapping that takes advantage of the ordered set of viable gene deletion mutants, which form a set of colinear markers covering almost every centimorgan of the Saccharomyces cerevisiae genome, and of the synthetic genetic array (SGA) system, which automates the construction of double mutants formed by mating and meiotic recombination. The Cbk1 kinase signaling pathway, which consists minimally of CBK1, MOB2, KIC1, HYM1, and TAO3 (PAG1), controls polarized morphogenesis and activation of the Ace2 transcription factor. Deletion mutations in the Cbk1 pathway genes are tolerated differently by common laboratory strains of S. cerevisiae, being viable in the W303 background but dead in the S288C background. Genetic analysis indicated that the lethality of Cbk1 pathway deletions in the S288C background was suppressed by a single allele specific to the W303 background. SGA mapping (SGAM) was used to locate this W303-specific suppressor to the SSD1 locus, which contains a known polymorphism that appears to compromise SSD1 function. This procedure should map any mutation, dominant or recessive, whose phenotype is epistatic to wild type, that is, a phenotype that can be scored from a mixed population of cells obtained by germination of both mutant and wild-type spores. In principle, SGAM should be applicable to the analysis of multigenic traits. Large-scale construction of ordered mutations in other model organisms would broaden the application of this approach.

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P1323HEMODIALYSIS TUNNELED CATHETER-RELATED BACTEREMIA: THIRTEEN-YEAR OBSERVATIONAL STUDY

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfaa142.p1323 ◽

2020 ◽

Vol 35 (Supplement_3) ◽

Author(s):

Marina Almenara Tejederas ◽

Wenceslao Aguilera Morales ◽

María Ángeles Rodríguez-Perez ◽

Salia Virxinia Pol Heres ◽

Mercedes Salgueira Lazo

Keyword(s):

Vascular Access ◽

Femoral Vein ◽

Insertion Site ◽

Clinical Signs ◽

Catheter Insertion ◽

High Rate ◽

Exclusion Criteria ◽

Gram Positive ◽

Management Protocol ◽

Tunneled Catheter

Abstract Background and Aims Tunneled catheter-related bacteremia (TCRB) is a common and severe cause of bacteremia among hemodialysis (HD)-dependent patients. TCRB have reported incidence of 0.5 to 5.5 events per 1000 catheter days and are associated with increased morbidity and death. The main objetive of our study is determinate the incidence of TCRB in our hospital and, secondarily, to analyze our microbiology, recurrence and reinfection rates. Method The study is an observational retrospective evaluation of medical records of patients in whom a TC for HD was implanted in the period from January 1, 2005, to December 31, 2018. The TC were implanted by nephrologists, following a preimplantation and management protocol agreed with the Infectious Diseases Unit. Patients were followed up from TC insertion until the study end date or first of recovery kidney function, kidney transplantation, transition to peritoneal dialysis or death. CRB definition was according Spanish Clinical Guidelines on Vascular Access for Haemodialysis: positive blood culture accompanied by fever or clinical signs of sepsis, without another posible site of infection. We recorded demographic, clinical and TC-related variables (conditions of catheter insertion, site of catheter insertion and duration of use, etc.). Exclusion criteria for our study were the lack of clinical follow-up due to belonging to a different hospital area. Results A total of 393 TC were implantated over a period of 13 years. After applying exclusion criteria, we investigated 341 TC implanted in 279 patients: 265 into the intern jugular vein, 71 into the subclavian and 5 in femoral vein. The mean age of the included patients was 63 (range 19-93 years). Fifty-one percent of catheter was implanted in male patients. Forty-six percent of the patients suffered from diabetes mellitus. In 55% of the cases, the cause of CT implantation was the difficulty of creating an internal vascular access. In total there were 91 CRB in 58 patients, with a rate of 0.48 infections per 1000 catheter days (figure 1), occurring at median 461 days (range 143-443 days) after catheter insertion. Within that group, 82.4% occurred after 6 months from the implementation of the CPT. Only 6 (6.59%) took place in the 30 days after implantation. Gram-positive organisms accounted of 85%, with a predominance of Staphylococcus epidermidis (47%) followed by Staphylococcus aureus (25%). A broad spectrum of Gram-negative bacteria accounts for 14% of patients. Nineteen TC were removed by CRB, with a rate of 5.5% of total functioning TC. CRB was the cause of death in 7 of the 279 patients (2.5%). During the study, 12 (13% of CRB) recurrences and 30 (32% of CRB) reinfections events have been identified. Conclusion The incidence of CRB in our population was found to be lower that previous studies. It usually appears in the long term, with Gram-positive germs as the most frequently involved. The temporality and low recurrence rate suggest that our protocol has been effective. The high rate of reinfection orients a certain individual predisposition to suffer from CRB. Identification of potential predicting risk factors could reduce the morbimortality of these patients.

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Uptake of Nanotitania by Gingival Epithelial Cells Promotes Inflammatory Response and Is Accelerated by Porphyromonas gingivalis Lipopolysaccharide: An In Vitro Study

International Journal of Molecular Sciences ◽

10.3390/ijms22158084 ◽

2021 ◽

Vol 22 (15) ◽

pp. 8084

Author(s):

Shiho Sugawara ◽

Taichi Ishikawa ◽

Shu Sato ◽

Hidemichi Kihara ◽

Masayuki Taira ◽

...

Keyword(s):

Epithelial Cells ◽

Porphyromonas Gingivalis ◽

Cell Model ◽

Quantitative Polymerase Chain Reaction ◽

High Rate ◽

Enzyme Linked Immunosorbent Assay ◽

Dependent Manner ◽

Periodontal Pathogens ◽

Gingival Epithelial Cells

Titanium is often used in the medical field and in dental implants due to its biocompatibility, but it has a high rate of leading to peri-implantitis, which progresses faster than periodontitis. Therefore, in the present study, the expression of cytokines from gingival epithelial cells by nanotitania was investigated, which is derived from titanium in the oral cavity, and the additional effect of Porphyromonas gingivalis (periodontopathic bacteria) lipopolysaccharide (PgLPS) was investigated. Ca9-22 cells were used as a gingival epithelial cell model and were cultured with nanotitania alone or with PgLPS. Cytokine expression was examined by reverse transcription-quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. In addition, cellular uptake of nanotitania was observed in scanning electron microscopy images. The expression of interleukin (IL)-6 and IL-8 significantly increased in Ca9-22 cells by nanotitania treatment alone, and the expression was further increased by the presence of PgLPS. Nanotitania was observed to phagocytose Ca9-22 cells in a dose- and time-dependent manner. Furthermore, when the expression of IL-11, related to bone resorption, was investigated, a significant increase was confirmed by stimulation with nanotitania alone. Therefore, nanotitania could be associated with the onset and exacerbation of peri-implantitis, and the presence of periodontal pathogens may worsen the condition. Further clinical reports are needed to confirm these preliminary results.

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