scholarly journals Super-Mendelian inheritance mediated by CRISPR/Cas9 in the female mouse germline

2018 ◽  
Author(s):  
Hannah A. Grunwald ◽  
Valentino M. Gantz ◽  
Gunnar Poplawski ◽  
Xiang-ru S. Xu ◽  
Ethan Bier ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Mendelian Inheritance ◽  
Early Embryo ◽  
Gene Drive ◽  
Dna Breaks ◽  
Homology Directed Repair ◽  
Double Strand Dna Breaks ◽  
Repair Mechanisms ◽  
Island Communities ◽  
Random Assortment

AbstractA gene drive biases the transmission of a particular allele of a gene such that it is inherited at a greater frequency than by random assortment. Recently, a highly efficient gene drive was developed in insects, which leverages the sequence-targeted DNA cleavage activity of CRISPR/Cas9 and endogenous homology directed repair mechanisms to convert heterozygous genotypes to homozygosity. If implemented in laboratory rodents, this powerful system would enable the rapid assembly of genotypes that involve multiple genes (e.g., to model multigenic human diseases). Such complex genetic models are currently precluded by time, cost, and a requirement for a large number of animals to obtain a few individuals of the desired genotype. However, the efficiency of a CRISPR/Cas9 gene drive system in mammals has not yet been determined. Here, we utilize an active genetic “CopyCat” element embedded in the mouse Tyrosinase gene to detect genotype conversions after Cas9 activity in the embryo and in the germline. Although Cas9 efficiently induces double strand DNA breaks in the early embryo and is therefore highly mutagenic, these breaks are not resolved by homology directed repair. However, when Cas9 expression is limited to the developing female germline, resulting double strand breaks are resolved by homology directed repair that copies the CopyCat allele from the donor to the receiver chromosome and leads to its super-Mendelian inheritance. These results demonstrate that the CRISPR/Cas9 gene drive mechanism can be implemented to simplify complex genetic crosses in laboratory mice and also contribute valuable data to the ongoing debate about applications to combat invasive rodent populations in island communities.

scholarly journals Double-strand DNA breaks recruit the centromeric histone CENP-A

2009 ◽  
Vol 106 (37) ◽  
pp. 15762-15767 ◽  
Author(s):  
Samantha G. Zeitlin ◽  
Norman M. Baker ◽  
Brian R. Chapados ◽  
Evi Soutoglou ◽  
Jean Y. J. Wang ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Dna Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Double Strand Dna Breaks ◽  
Histone H3 Variant ◽  
Radiation Induced ◽  
Non Homologous End Joining

The histone H3 variant CENP-A is required for epigenetic specification of centromere identity through a loading mechanism independent of DNA sequence. Using multiphoton absorption and DNA cleavage at unique sites by I-SceI endonuclease, we demonstrate that CENP-A is rapidly recruited to double-strand breaks in DNA, along with three components (CENP-N, CENP-T, and CENP-U) associated with CENP-A at centromeres. The centromere-targeting domain of CENP-A is both necessary and sufficient for recruitment to double-strand breaks. CENP-A accumulation at DNA breaks is enhanced by active non-homologous end-joining but does not require DNA-PKcs or Ligase IV, and is independent of H2AX. Thus, induction of a double-strand break is sufficient to recruit CENP-A in human and mouse cells. Finally, since cell survival after radiation-induced DNA damage correlates with CENP-A expression level, we propose that CENP-A may have a function in DNA repair.

Editing livestock genomes with site-specific nucleases

2014 ◽  
Vol 26 (1) ◽  
pp. 74 ◽  
Author(s):  
Daniel F. Carlson ◽  
Wenfang Tan ◽  
Perry B. Hackett ◽  
Scott C. Fahrenkrug
Keyword(s):  
Genetic Alterations ◽  
Large Animal ◽  
Dna Breaks ◽  
Transcription Activator ◽  
End Joining ◽  
Site Specific ◽  
Homology Directed Repair ◽  
Double Strand Dna Breaks ◽  
Non Homologous End Joining ◽  
Inactive Genes

Over the past 5 years there has been a major transformation in our ability to precisely manipulate the genomes of animals. Efficiencies of introducing precise genetic alterations in large animal genomes have improved 100 000-fold due to a succession of site-specific nucleases that introduce double-strand DNA breaks with a specificity of 10–9. Herein we describe our applications of site-specific nucleases, especially transcription activator-like effector nucleases, to engineer specific alterations in the genomes of pigs and cows. We can introduce variable changes mediated by non-homologous end joining of DNA breaks to inactive genes. Alternatively, using homology-directed repair, we have introduced specific changes that support either precise alterations in a gene’s encoded polypeptide, elimination of the gene or replacement by another unrelated DNA sequence. Depending on the gene and the mutation, we can achieve 10%–50% effective rates of precise mutations. Applications of the new precision genetics are extensive. Livestock now can be engineered with selected phenotypes that will augment their value and adaption to variable ecosystems. In addition, animals can be engineered to specifically mimic human diseases and disorders, which will accelerate the production of reliable drugs and devices. Moreover, animals can be engineered to become better providers of biomaterials used in the medical treatment of diseases and disorders.

scholarly journals ERCC1-XPF Interacts with Topoisomerase IIβ to Facilitate the Repair of Activity-induced DNA Breaks

2020 ◽  
Author(s):  
Georgia Chatzinikolaou ◽  
Kalliopi Stratigi ◽  
Kyriacos Agathangelou ◽  
Maria Tsekrekou ◽  
Evi Goulielmaki ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Genotoxic Stress ◽  
Gene Promoters ◽  
Dna Breaks ◽  
Double Strand Dna Breaks ◽  
Genome Wide ◽  
Human Disorders ◽  
Or Gene ◽  
Type Ii Dna Topoisomerases

AbstractType II DNA Topoisomerases (TOP II) generate transient double-strand DNA breaks (DSBs) to resolve topological constraints during transcription. Using genome-wide mapping of DSBs and functional genomics approaches, we show that, in the absence of exogenous genotoxic stress, transcription leads to DSB accumulation and to the recruitment of the structure-specific ERCC1-XPF endonuclease on active gene promoters. Instead, we find that the complex is released from regulatory or gene body elements in UV-irradiated cells. Abrogation of ERCC1 or re-ligation blockage of TOP II-mediated DSBs aggravates the accumulation of transcription-associated γH2Ax and 53BP1 foci, which dissolve when TOP II-mediated DNA cleavage is inhibited. An in vivo biotinylation tagging strategy coupled to a high-throughput proteomics approach reveals that ERCC1-XPF interacts with TOP IIβ and the CTCF/cohesin complex, which co-localize with the heterodimer on DSBs. Together; our findings provide a rational explanation for the remarkable clinical heterogeneity seen in human disorders with ERCC1-XPF defects.

scholarly journals Analysis of off-target effects in CRISPR-based gene drives in the human malaria mosquito

2021 ◽  
Vol 118 (22) ◽  
pp. e2004838117
Author(s):  
William T. Garrood ◽  
Nace Kranjc ◽  
Karl Petri ◽  
Daniel Y. Kim ◽  
Jimmy A. Guo ◽  
...  
Keyword(s):  
Mendelian Inheritance ◽  
Gene Drive ◽  
Guide Rna ◽  
Human Malaria ◽  
Homology Directed Repair ◽  
Cas9 Nuclease ◽  
Set Up ◽  
Gene Drives ◽  
Target Effects ◽  
Important Milestone

CRISPR-Cas9 nuclease-based gene drives have been developed toward the aim of control of the human malaria vector Anopheles gambiae. Gene drives are based on an active source of Cas9 nuclease in the germline that promotes super-Mendelian inheritance of the transgene by homology-directed repair (“homing”). Understanding whether CRISPR-induced off-target mutations are generated in Anopheles mosquitoes is an important aspect of risk assessment before any potential field release of this technology. We compared the frequencies and the propensity of off-target events to occur in four different gene-drive strains, including a deliberately promiscuous set-up, using a nongermline restricted promoter for SpCas9 and a guide RNA with many closely related sites (two or more mismatches) across the mosquito genome. Under this scenario we observed off-target mutations at frequencies no greater than 1.42%. We witnessed no evidence that CRISPR-induced off-target mutations were able to accumulate (or drive) in a mosquito population, despite multiple generations’ exposure to the CRISPR-Cas9 nuclease construct. Furthermore, judicious design of the guide RNA used for homing of the CRISPR construct, combined with tight temporal constriction of Cas9 expression to the germline, rendered off-target mutations undetectable. The findings of this study represent an important milestone for the understanding and managing of CRISPR-Cas9 specificity in mosquitoes, and demonstrates that CRISPR off-target editing in the context of a mosquito gene drive can be reduced to minimal levels.

scholarly journals Topoisomerase 2b induces DNA breaks to regulate human papillomavirus replication

2021 ◽  
Author(s):  
Paul Kaminski ◽  
Shiyuan Hong ◽  
Takeyuki Kono ◽  
Paul Hoover ◽  
Laimonis A. Laimins
Keyword(s):  
Dna Cleavage ◽  
Genome Replication ◽  
Dna Breaks ◽  
Double Strand Dna Breaks ◽  
Damage Repair ◽  
High Risk Hpv ◽  
Strand Passage ◽  
Covalent Intermediate ◽  
Do So

Topoisomerases regulate higher order chromatin structures through the transient breaking and re-ligating of one or both strands of the phosphodiester backbone of duplex DNA. TOP2b is a type II topoisomerase that induces double strand DNA breaks at topological-associated domains (TADS) to relieve torsional stress arising during transcription or replication. TADS are anchored by CTCF and SMC1 cohesin proteins in complexes with TOP2b. Upon DNA cleavage a covalent intermediate DNA-TOP2b (TOP2bcc) is transiently generated to allow for strand passage. The tyrosyl-DNA phosphodiesterase TDP2 can resolve TOP2bcc but failure to do so quickly can lead to long-lasting DNA breaks. Given the role of CTCF/SMC1 proteins in the HPV life cycle we investigated if TOP2b proteins contribute to HPV pathogenesis. Our studies demonstrated that levels of both TOP2b and TDP2 were substantially increased in cells with high risk HPV genomes and this correlated with high amounts of DNA breaks. Knockdown of TOP2b with shRNAs reduced DNA breaks by over 50% as determined through COMET assays.  Furthermore this correlated with substantially reduced formation of repair foci such as gH2AX, pCHK1 and pSMC1 indicative of impaired activation of DNA damage repair pathways. Importantly, knockdown of TOP2b also blocked HPV genome replication. Our previous studies demonstrated that CTCF /SMC1 factors associate with HPV genomes at sites in the late regions of HPV31 and these correspond to regions that also bind TOP2b. This study identifies TOP2b as responsible for enhanced levels of DNA breaks in HPV positive cells and as a regulator of viral replication.

scholarly journals Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Steven Lin ◽  
Brett T Staahl ◽  
Ravi K Alla ◽  
Jennifer A Doudna
Keyword(s):  
Genome Engineering ◽  
High Efficiency ◽  
Embryonic Stem ◽  
Human Cells ◽  
Dna Breaks ◽  
Site Specific ◽  
Guide Rna ◽  
Homology Directed Repair ◽  
Double Strand Dna Breaks ◽  
Cell Mortality

The CRISPR/Cas9 system is a robust genome editing technology that works in human cells, animals and plants based on the RNA-programmed DNA cleaving activity of the Cas9 enzyme. Building on previous work (<xref ref-type="bibr" rid="bib13">Jinek et al., 2013</xref>), we show here that new genetic information can be introduced site-specifically and with high efficiency by homology-directed repair (HDR) of Cas9-induced site-specific double-strand DNA breaks using timed delivery of Cas9-guide RNA ribonucleoprotein (RNP) complexes. Cas9 RNP-mediated HDR in HEK293T, human primary neonatal fibroblast and human embryonic stem cells was increased dramatically relative to experiments in unsynchronized cells, with rates of HDR up to 38% observed in HEK293T cells. Sequencing of on- and potential off-target sites showed that editing occurred with high fidelity, while cell mortality was minimized. This approach provides a simple and highly effective strategy for enhancing site-specific genome engineering in both transformed and primary human cells.

scholarly journals Timed inhibition of CDC7 increases CRISPR-Cas9 mediated templated repair

2018 ◽  
Author(s):  
Beeke Wienert ◽  
Sharon J Feng ◽  
Melissa Locke ◽  
David N Nguyen ◽  
Stacia K Wyman ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Phase Cell ◽  
Dna Breaks ◽  
Gene Modification ◽  
Individual Gene ◽  
Homology Directed Repair ◽  
Cycle Arrest ◽  
Double Strand Dna Breaks ◽  
Repair Pathways ◽  
Cell Division Cycle 7

Repair of double strand DNA breaks (DSBs) can result in gene disruption or precise gene modification via homology directed repair (HDR) from a templating donor DNA. During genome editing, altering cellular responses to DSBs may be an effective strategy to rebalance editing outcomes towards HDR and away from other repair pathways. To identify factors that regulate HDR from a double-stranded DNA donor (dsDonor), we utilized a pooled screen to define the consequences of thousands of individual gene knockdowns during Cas9-initiated HDR from a double strand plasmid donor. We find that templated dsDonor repair pathways are mostly genetically distinct from single strand donor DNA (ssDonor) repair but share aspects that include dependency upon the Fanconi Anemia (FA) pathway. We also identified several factors whose knockdown increases HDR and thus act as repressors of gene modification. Screening available small molecule inhibitors of these repressors revealed that the cell division cycle 7-related protein kinase (CDC7) inhibitor XL413 increases the efficiency of HDR by 2-3 fold in many contexts, including primary T-cells. XL413 stimulates HDR through cell cycle regulation, inducing an early S-phase cell cycle arrest that, to the best of our knowledge, is uncharacterized for Cas9-induced HDR. We anticipate that XL413 and other such rationally developed inhibitors will be useful tools for boosting the efficiency of gene modification.

scholarly journals Multiple loci of small effect confer wide variability in efficiency and resistance rate of CRISPR gene drive

2018 ◽  
Author(s):  
Jackson Champer ◽  
Zhaoxin Wen ◽  
Anisha Luthra ◽  
Riona Reeves ◽  
Joan Chung ◽  
...  
Keyword(s):  
Genome Wide Association Study ◽  
Early Embryo ◽  
Resistance Rate ◽  
Pathogen Transmission ◽  
Gene Drive ◽  
Natural Genetic Variation ◽  
Homology Directed Repair ◽  
Conversion Rates ◽  
A Genome ◽  
Gene Drives

ABSTRACTGene drives could allow for control of vector-borne diseases by directly suppressing vector populations or spreading genetic payloads designed to reduce pathogen transmission. CRISPR homing gene drives work by cleaving wild-type alleles, which are then converted to drive alleles by homology-directed repair, increasing the frequency of the drive in a population. However, resistance alleles can form when end-joining repair takes place in lieu of homology-directed repair. Such alleles cannot be converted to drive alleles, which would halt the spread of a drive through a population. To investigate the effects of natural genetic variation on resistance formation, we developed a CRISPR homing gene drive in Drosophila melanogaster and crossed it into the genetically diverse Drosophila Genetic Reference Panel (DGRP) lines, measuring several performance parameters. Most strikingly, resistance allele formation post-fertilization in the early embryo ranged from 7% to 79% among lines and averaged 42±18%. We performed a Genome-Wide Association Study (GWAS) using our results in the DGRP lines and found that the resistance and conversion rates were polygenic, with several genetic polymorphisms showing relatively weak association. RNAi knockdown of several of these genes confirmed their effect, but their small effect sizes implies that their manipulation will yield only modest improvements to the efficacy of gene drives.

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