scholarly journals A method to estimate the frequency of chromosomal rearrangements induced by CRISPR/Cas9 multiplexing in Drosophila

2019 ◽  
Author(s):  
William A. Ng ◽  
Bruce H. Reed
Keyword(s):  
High Frequency ◽  
Pericentric Inversion ◽  
Target Genes ◽  
Chromosomal Rearrangements ◽  
Chromosome Analysis ◽  
Selection Method ◽  
Salivary Gland Polytene Chromosome ◽  
Target Sites ◽  
Drosophila Line ◽  
Female Germline

AbstractUsing CRISPR/Cas9 to simultaneously induce mutations in two or more target genes, commonly referred to as multiplexing, may result in chromosomal rearrangements such as inversions or translocations. While this may be undesirable in some contexts, the ability to recover chromosomal rearrangements targeted to specific sites in the genome is potentially a powerful tool. Before developing such tools, however, it is first important to measure the frequency with which chromosome rearrangements are induced by CRISPR/Cas9 multiplexing. To do this, we have developed a self-selecting screening system using a Drosophila line that carries an autosomal pericentric inversion in what is known as the autosynaptic form. All progeny of normal females crossed to males of this autosynaptic stock are lethal due to excessive aneuploidy. If an inversion is induced within the female germline, and if it is analogous to the inversion in the male autosynaptic line, then it is possible to recover progeny in which aneuploidy is reduced and viability is restored. Using this self-selection method, we screened 130 females and recovered one new autosynaptic element. Salivary gland polytene chromosome analysis, PCR, and sequencing confirmed the recovery of a breakpoint induced precisely between the two sgRNA target sites. Overall, we demonstrate that CRISPR/Cas9 multiplexing can induce chromosomal rearrangements in Drosophila. Also, in using this particular system, the recovery of chromosomal rearrangements was not a high frequency event.

scholarly journals Global Analysis of the Genetic Variations in miRNA-Targeted Sites and Their Correlations With Agronomic Traits in Rapeseed

2021 ◽  
Vol 12 ◽  
Author(s):  
Pengfei Xu ◽  
Yantao Zhu ◽  
Yanfeng Zhang ◽  
Jianxia Jiang ◽  
Liyong Yang ◽  
...  
Keyword(s):  
Mirna Target ◽  
Target Genes ◽  
Rare Variants ◽  
Agronomic Traits ◽  
Global Analysis ◽  
Variant Calling ◽  
Genetic Variations ◽  
Nucleotide Polymorphisms ◽  
A Genome ◽  
Target Sites

MicroRNAs (miRNAs) and their target genes play vital roles in crops. However, the genetic variations in miRNA-targeted sites that affect miRNA cleavage efficiency and their correlations with agronomic traits in crops remain unexplored. On the basis of a genome-wide DNA re-sequencing of 210 elite rapeseed (Brassica napus) accessions, we identified the single nucleotide polymorphisms (SNPs) and insertions/deletions (INDELs) in miRNA-targeted sites complementary to miRNAs. Variant calling revealed 7.14 million SNPs and 2.89 million INDELs throughout the genomes of 210 rapeseed accessions. Furthermore, we detected 330 SNPs and 79 INDELs in 357 miRNA target sites, of which 33.50% were rare variants. We also analyzed the correlation between the genetic variations in miRNA target sites and 12 rapeseed agronomic traits. Eleven SNPs in miRNA target sites were significantly correlated with phenotypes in three consecutive years. More specifically, three correlated SNPs within the miRNA-binding regions of BnSPL9-3, BnSPL13-2, and BnCUC1-2 were in the loci associated with the branch angle, seed weight, and silique number, respectively; expression profiling suggested that the variation at these 3 miRNA target sites significantly affected the expression level of the corresponding target genes. Taken together, the results of this study provide researchers and breeders with a global view of the genetic variations in miRNA-targeted sites in rapeseed and reveal the potential effects of these genetic variations on elite agronomic traits.

scholarly journals Differentiating Drosophila female germ cells initiate Polycomb silencing by regulating PRC2-interacting proteins

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Steven Z DeLuca ◽  
Megha Ghildiyal ◽  
Liang-Yu Pang ◽  
Allan C Spradling
Keyword(s):  
Interacting Proteins ◽  
Nurse Cells ◽  
Germline Stem Cells ◽  
Developmentally Regulated ◽  
Canonical Distribution ◽  
Polycomb Repressive Complex 2 ◽  
Animal Development ◽  
Target Sites ◽  
Female Germline ◽  
Inactive Chromatin

Polycomb silencing represses gene expression and provides a molecular memory of chromatin state that is essential for animal development. We show that Drosophila female germline stem cells (GSCs) provide a powerful system for studying Polycomb silencing. GSCs have a non-canonical distribution of PRC2 activity and lack silenced chromatin like embryonic progenitors. As GSC daughters differentiate into nurse cells and oocytes, nurse cells, like embryonic somatic cells, silence genes in traditional Polycomb domains and in generally inactive chromatin. Developmentally controlled expression of two Polycomb repressive complex 2 (PRC2)-interacting proteins, Pcl and Scm, initiate silencing during differentiation. In GSCs, abundant Pcl inhibits PRC2-dependent silencing globally, while in nurse cells Pcl declines and newly induced Scm concentrates PRC2 activity on traditional Polycomb domains. Our results suggest that PRC2-dependent silencing is developmentally regulated by accessory proteins that either increase the concentration of PRC2 at target sites or inhibit the rate that PRC2 samples chromatin.

Hyperdiploidy and chromosomal rearrangements define the anaplastic variant of Wilms' tumor.

1986 ◽  
Vol 4 (6) ◽  
pp. 975-981 ◽  
Author(s):  
E C Douglass ◽  
A T Look ◽  
B Webber ◽  
D Parham ◽  
J A Wilimas ◽  
...  
Keyword(s):  
Dna Content ◽  
Wilms Tumor ◽  
Chromosomal Rearrangements ◽  
Chromosome Analysis ◽  
Histologic Subtype ◽  
Flow Cytometric ◽  
Kaplan Meier ◽  
Flow Cytometric Measurement ◽  
Flow Cytometric Study ◽  
Diploid Cells

Flow cytometric measurement of the DNA content of Wilms' tumor cells revealed a striking correspondence with the histologic subtype and treatment outcome. In the 48 cases studied, a hyperdiploid DNA content ranging from 1.7 to 3.2 times the result for normal diploid cells distinguished all but one of the ten anaplastic tumors. Lower values, from 1.0 to 1.4 times the diploid DNA content, characterized the nonanaplastic specimens. By Kaplan-Meier analysis, the probability of achieving 3 years of relapse-free survival was significantly lower in the group with higher DNA content (0.42 v 0.87, P less than .01). Analysis of banded chromosomes for a subset of 22 patients contributed important information beyond the flow cytometric study. Cases of anaplasia associated with poorer responses to therapy showed numerous complex translocations, whereas all others lacked such changes. By combining flow cytometric techniques and conventional methods of chromosome analysis, it should be possible to identify those patients with Wilms' tumor who are most likely to fail therapy. The biologic implication of these findings is that the development of clinical drug resistance in Wilms' tumor is a result of the genetic instability of the malignant clone.

scholarly journals Snake W Sex Chromosome: The Shadow of Ancestral Amniote Super-Sex Chromosome

Cells ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 2386
Author(s):  
Worapong Singchat ◽  
Syed Farhan Ahmad ◽  
Nararat Laopichienpong ◽  
Aorarat Suntronpong ◽  
Thitipong Panthum ◽  
...  
Keyword(s):  
Sex Chromosomes ◽  
Evolutionary Dynamics ◽  
Sex Chromosome ◽  
Chromosomal Rearrangements ◽  
Chromosome Analysis ◽  
Chromosome Conformation ◽  
Principal Mechanism ◽  
Squamate Reptiles ◽  
Genomic Landscape ◽  
Heteromorphic Sex Chromosomes

Heteromorphic sex chromosomes, particularly the ZZ/ZW sex chromosome system of birds and some reptiles, undergo evolutionary dynamics distinct from those of autosomes. The W sex chromosome is a unique karyological member of this heteromorphic pair, which has been extensively studied in snakes to explore the origin, evolution, and genetic diversity of amniote sex chromosomes. The snake W sex chromosome offers a fascinating model system to elucidate ancestral trajectories that have resulted in genetic divergence of amniote sex chromosomes. Although the principal mechanism driving evolution of the amniote sex chromosome remains obscure, an emerging hypothesis, supported by studies of W sex chromosomes of squamate reptiles and snakes, suggests that sex chromosomes share varied genomic blocks across several amniote lineages. This implies the possible split of an ancestral super-sex chromosome via chromosomal rearrangements. We review the major findings pertaining to sex chromosomal profiles in amniotes and discuss the evolution of an ancestral super-sex chromosome by collating recent evidence sourced mainly from the snake W sex chromosome analysis. We highlight the role of repeat-mediated sex chromosome conformation and present a genomic landscape of snake Z and W chromosomes, which reveals the relative abundance of major repeats, and identifies the expansion of certain transposable elements. The latest revolution in chromosomics, i.e., complete telomere-to-telomere assembly, offers mechanistic insights into the evolutionary origin of sex chromosomes.

scholarly journals Chromosomal Evolution in the Lizard Genus Varanus (Reptilia)

1975 ◽  
Vol 28 (1) ◽  
pp. 89 ◽  
Author(s):  
Max Kinga ◽  
Dennis King
Keyword(s):  
Chromosome Number ◽  
Pericentric Inversion ◽  
Sex Chromosome ◽  
Chromosomal Rearrangements ◽  
Chromosomal Evolution ◽  
Size Groups ◽  
Sex Chromosome System

The karyotypes have been determined of 16 of the 32 species of the genus Varanus, including animals from Africa, Israel, Malaya and Australia. A constant chromosome number of 2n = 40 was observed. The karyotype is divided into eight pairs of large chromosomes and 12 pairs of microchromosomes. A series of chromosomal rearrangements have become established in both size groups of the karyotype and are restricted to centromere shifts, probably caused by pericentric inversion. Species could be placed in one of six distinct karyotype groups which are differentiated by these rearrangements and whose grouping does not always correspond with the current taxonomy. An unusual sex chromosome system of the ZZjZW type was present in a number of the species examined.

scholarly journals Microhomologies are prevalent at Cas9-induced larger deletions

2019 ◽  
Vol 47 (14) ◽  
pp. 7402-7417 ◽  
Author(s):  
Dominic D G Owens ◽  
Adam Caulder ◽  
Vincent Frontera ◽  
Joe R Harman ◽  
Alasdair J Allan ◽  
...  
Keyword(s):  
Genome Editing ◽  
Biomedical Research ◽  
High Frequency ◽  
Cultured Cells ◽  
Mouse Embryos ◽  
Deletion Breakpoint ◽  
End Joining ◽  
Crispr System ◽  
Cas9 Nuclease ◽  
Target Sites

Abstract The CRISPR system is widely used in genome editing for biomedical research. Here, using either dual paired Cas9D10A nickases or paired Cas9 nuclease we characterize unintended larger deletions at on-target sites that frequently evade common genotyping practices. We found that unintended larger deletions are prevalent at multiple distinct loci on different chromosomes, in cultured cells and mouse embryos alike. We observed a high frequency of microhomologies at larger deletion breakpoint junctions, suggesting the involvement of microhomology-mediated end joining in their generation. In populations of edited cells, the distribution of larger deletion sizes is dependent on proximity to sgRNAs and cannot be predicted by microhomology sequences alone.

scholarly journals The pancreatic islet factor STF-1 binds cooperatively with Pbx to a regulatory element in the somatostatin promoter: importance of the FPWMK motif and of the homeodomain.

1995 ◽  
Vol 15 (12) ◽  
pp. 7091-7097 ◽  
Author(s):  
B Peers ◽  
S Sharma ◽  
T Johnson ◽  
M Kamps ◽  
M Montminy
Keyword(s):  
Target Genes ◽  
Gene Selection ◽  
Regulatory Element ◽  
Regulatory Elements ◽  
Cooperative Binding ◽  
Homeodomain Protein ◽  
Homeodomain Proteins ◽  
Target Sites

A number of homeodomain proteins have been shown to regulate cellular development by stimulating the transcription of specific target genes. In contrast to their distinct activities in vivo, however, most homeodomain proteins bind indiscriminately to potential target sites in vitro, suggesting the involvement of cofactors which specify target site selection. One such cofactor, termed extradenticle, has been shown to influence segmental morphogenesis in Drosophila melanogaster by binding cooperatively with certain homeodomain proteins to target regulatory elements. Here we demonstrate that STF-1, an orphan homeodomain protein required for pancreatic development in mammals, binds cooperatively to DNA with Pbx, the mammalian homolog of extradenticle. Cooperative binding with Pbx requires a pentapeptide motif (FPWMK) which is well conserved among a large subset of homeodomain proteins. The FPMWK motif is not sufficient to confer Pbx cooperativity on other homeodomain proteins, however; the N-terminal arm of the STF-1 homeodomain is also essential. As cooperative binding with Pbx occurs on only a subset of potential STF-1 target sites, our results suggest that Pbx may specify target gene selection in the developing pancreas by forming heterodimeric complexes with STF-1.

CHROMOSOME ANALYSIS OF SCHISTOSOMA RODHAINI (TREMATODA: SCHISTOSOMATIDAE)

1980 ◽  
Vol 22 (1) ◽  
pp. 143-147 ◽  
Author(s):  
Kristine H. Atkinson
Keyword(s):  
Pericentric Inversion ◽  
Chromosome Pair ◽  
Mitotic Chromosome ◽  
Nucleolar Organizer ◽  
Chromosome Analysis ◽  
Host Tissue ◽  
Similar Species ◽  
Group I ◽  
Field Identification ◽  
Hypotonic Treatment

The chromosome number of Schistosoma rodhaini Brumpt is 2n = 16, with apparent sexual dimorphism quantifiable in chromosome pair no. 2. A method for dissociating host tissue coupled with hypotonic treatment yields permanent mitotic chromosome spreads with very defined centromere regions. The karyotype of S. rodhaini is very similar to that of its sibling species, S. mansoni Sambon, except there is a pericentric inversion in the S. rodhaini nucleolar organizer chromosome pair, S. rodhaini does not have any satellited chromosomes, and S. rodhaini has slightly shorter short arms of the group I chromosomes than S. mansoni. This distinction of chromosomes of similar species of schistosomes will be important for field identification of parasites and in elucidating the evolution of the schistosomes.

scholarly journals A Frequency Selection Method Based on the Pole Characteristics

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
Songyan Yang ◽  
Weibo Deng ◽  
Qiang Yang ◽  
Guangxin Wu ◽  
Ying Suo
Keyword(s):  
Frequency Band ◽  
Incident Wave ◽  
High Frequency ◽  
Recognition Rate ◽  
Selection Method ◽  
High Frequency Band ◽  
Optimal Frequency ◽  
Frequency Selection ◽  
Average Recognition Rate

Due to the heavy jamming band of high frequency, frequency selecting strategies are serious issues for the system designed to achieve its best performance. Pole is independent of the direction and polarization of the incident wave, but the residue corresponding to the pole is related to the direction and polarization of the incident wave. And the value of residue is proportional to the value of the pole. This paper chooses the frequency which can maximize the residue in the high-frequency band as the optimal frequency for accurate extraction. The simulation result of a large number of ship targets shows remarkable rise in average recognition rate by using this method, compared with the average recognition rate of randomly selected frequency.

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