scholarly journals Heterochromatin delays CRISPR-Cas9 mutagenesis but does not influence repair outcome

2018 ◽  
Author(s):  
Eirini M Kallimasioti-Pazi ◽  
Keerthi Thelakkad Chathoth ◽  
Gillian C Taylor ◽  
Alison Meynert ◽  
Tracy Ballinger ◽  
...  
Keyword(s):  
Genome Editing ◽  
Imprinted Genes ◽  
Induced Mutations ◽  
Permeable Barrier ◽  
Homology Directed Repair ◽  
Confounding Variables ◽  
Experimental Parameters ◽  
Allele Specific ◽  
And Function ◽  
Mutagenic Dna Repair

AbstractCRISPR-Cas9 genome editing occurs in the context of chromatin, which is heterogeneous in structure and function across the genome. Chromatin heterogeneity is thought to affect genome editing efficiency, but this has been challenging to quantify due to the presence of confounding variables. Here, we develop a method that exploits the allele-specific chromatin status of imprinted genes in order to address this problem. Because maternal and paternal alleles of imprinted genes have identical DNA sequence and are situated in the same nucleus, allele-specific differences in the frequency and spectrum of Cas9-induced mutations can be attributed unequivocally to epigenetic mechanisms. We found that heterochromatin can impede mutagenesis, but to a degree that depends on other key experimental parameters. Mutagenesis was impeded by up to 7-fold when Cas9 exposure was brief and when intracellular Cas9 expression was low. Surprisingly, the outcome of mutagenic DNA repair was independent of chromatin state, with similar efficiencies of homology directed repair and deletion spectra on maternal and paternal chromosomes. Combined, our data show that heterochromatin imposes a permeable barrier that influences the kinetics, but not the endpoint of CRISPR-Cas9 genome editing, and suggest that therapeutic applications involving low-level Cas9 exposure will be particularly affected by chromatin status.

Allele-specific genome editing using CRISPR-Cas9 causes off-target mutations in diploid yeast

2018 ◽  
Author(s):  
Arthur R. Gorter de Vries ◽  
Lucas G. F. Couwenberg ◽  
Marcel van den Broek ◽  
Pilar de la Torre Cortés ◽  
Jolanda ter Horst ◽  
...  
Keyword(s):  
Genome Editing ◽  
Loss Of Heterozygosity ◽  
Genetic Modification ◽  
Large Scale ◽  
Gene Editing ◽  
Yeast Genome ◽  
Dna Double Strand Breaks ◽  
Homology Directed Repair ◽  
Eukaryotic Gene ◽  
Allele Specific

ABSTRACTTargeted DNA double-strand breaks (DSBs) with CRISPR-Cas9 have revolutionized genetic modification by enabling efficient genome editing in a broad range of eukaryotic systems. Accurate gene editing is possible with near-perfect efficiency in haploid or (predominantly) homozygous genomes. However, genomes exhibiting polyploidy and/or high degrees of heterozygosity are less amenable to genetic modification. Here, we report an up to 99-fold lower gene editing efficiency when editing individual heterozygous loci in the yeast genome. Moreover, Cas9-mediated introduction of a DSB resulted in large scale loss of heterozygosity affecting DNA regions up to 360 kb that resulted in introduction of nearly 1700 off-target mutations, due to replacement of sequences on the targeted chromosome by corresponding sequences from its non-targeted homolog. The observed patterns of loss of heterozygosity were consistent with homology directed repair. The extent and frequency of loss of heterozygosity represent a novel mutagenic side-effect of Cas9-mediated genome editing, which would have to be taken into account in eukaryotic gene editing. In addition to contributing to the limited genetic amenability of heterozygous yeasts, Cas9-mediated loss of heterozygosity could be particularly deleterious for human gene therapy, as loss of heterozygous functional copies of anti-proliferative and pro-apoptotic genes is a known path to cancer.

Single Nucleotide Substitutions Effectively Block Cas9 and Allow for Scarless Genome Editing in Caenorhabditis elegans

2021 ◽  
Author(s):  
Jeffrey C Medley ◽  
Shilpa Hebbar ◽  
Joel T Sydzyik ◽  
Anna Y. Zinovyeva
Keyword(s):  
Caenorhabditis Elegans ◽  
Genome Editing ◽  
Dna Breaks ◽  
Nucleotide Substitutions ◽  
Paternal Genome ◽  
Single Nucleotide ◽  
C Elegans ◽  
Homology Directed Repair ◽  
Maternal Genome ◽  
Guide Sequence

In Caenorhabditis elegans, germline injection of Cas9 complexes is reliably used to achieve genome editing through homology-directed repair of Cas9-generated DNA breaks. To prevent Cas9 from targeting repaired DNA, additional blocking mutations are often incorporated into homologous repair templates. Cas9 can be blocked either by mutating the PAM sequence that is essential for Cas9 activity or by mutating the guide sequence that targets Cas9 to a specific genomic location. However, it is unclear how many nucleotides within the guide sequence should be mutated, since Cas9 can recognize off-target sequences that are imperfectly paired to its guide. In this study, we examined whether single-nucleotide substitutions within the guide sequence are sufficient to block Cas9 and allow for efficient genome editing. We show that a single mismatch within the guide sequence effectively blocks Cas9 and allows for recovery of edited animals. Surprisingly, we found that a low rate of edited animals can be recovered without introducing any blocking mutations, suggesting a temporal block to Cas9 activity in C. elegans. Furthermore, we show that the maternal genome of hermaphrodite animals is preferentially edited over the paternal genome. We demonstrate that maternally provided haplotypes can be selected using balancer chromosomes and propose a method of mutant isolation that greatly reduces screening efforts post-injection. Collectively, our findings expand the repertoire of genome editing strategies in C. elegans and demonstrate that extraneous blocking mutations are not required to recover edited animals when the desired mutation is located within the guide sequence.

Genes Regulating Immune Response and Amelogenesis Interact in Increasing the Susceptibility to Molar-Incisor Hypomineralization

2018 ◽  
Vol 53 (2) ◽  
pp. 217-227 ◽  
Author(s):  
Diego Girotto Bussaneli ◽  
Manuel Restrepo ◽  
Camila Maria Bullio Fragelli ◽  
Lourdes Santos-Pinto ◽  
Fabiano Jeremias ◽  
...  
Keyword(s):  
Immune Response ◽  
Gene Interactions ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Nuclear Families ◽  
Molar Incisor Hypomineralization ◽  
Inflammatory Stimuli ◽  
Allele Specific ◽  
Family Based ◽  
And Function

Ameloblasts are sensitive cells whose metabolism and function may be affected by inflammatory stimuli. The aim of this study was to evaluate the possible association between polymorphisms in immune response-related genes and molar-incisor hypomineralization (MIH), and their interaction with polymorphisms in amelogenesis-related genes. DNA samples were obtained from 101 nuclear families that had at least 1 MIH-affected child. Eleven single-nucleotide polymorphisms (SNPs) were investigated in immune response genes using TaqMan® technology allele-specific probes. A transmission disequilibrium test was performed to verify overtransmission of alleles in all MIH families, as well as in families only with mild or severe MIH-affected children. Gene-gene interactions between the immune-related and amelogenesis-related polymorphisms were analyzed by determining whether alleles of those genes were transmitted from heterozygous parents more often in association than individually with MIH-affected children. In severe cases of MIH, significant results were observed for rs10733708 (TGFBR1, OR = 3.5, 95% CI = 1.1–10.6). Statistical evidence for gene-gene interactions between rs6654939 (AMELX) and the SNPs rs2070874 (IL4), rs2275913 (IL17A), rs1800872 (IL10), rs1800587 (IL1A), and rs3771300 (STAT1) was observed. The rs2070874 SNP (IL4) was also significantly overtransmitted from heterozygous parents with the rs7526319 (TUFT1) and the rs2355767 (BMP2) SNPs, suggesting a synergistic effect of the transmission of these alleles with susceptibility to MIH. This family-based study demonstrated an association between variation in TGFBR1 and MIH. Moreover, the polymorphisms in immune response and amelogenesis genes may have an additive effect on the risk of developing MIH.

scholarly journals Direct regulation of Arp2/3 complex activity and function by the actin binding protein coronin

2002 ◽  
Vol 159 (6) ◽  
pp. 993-1004 ◽  
Author(s):  
Christine L. Humphries ◽  
Heath I. Balcer ◽  
Jessica L. D'Agostino ◽  
Barbara Winsor ◽  
David G. Drubin ◽  
...  
Keyword(s):  
Binding Protein ◽  
Coiled Coil ◽  
Actin Binding ◽  
Complex Activity ◽  
Actin Binding Protein ◽  
Coiled Coil Domain ◽  
Allele Specific ◽  
And Function

Mechanisms for activating the actin-related protein 2/3 (Arp2/3) complex have been the focus of many recent studies. Here, we identify a novel mode of Arp2/3 complex regulation mediated by the highly conserved actin binding protein coronin. Yeast coronin (Crn1) physically associates with the Arp2/3 complex and inhibits WA- and Abp1-activated actin nucleation in vitro. The inhibition occurs specifically in the absence of preformed actin filaments, suggesting that Crn1 may restrict Arp2/3 complex activity to the sides of filaments. The inhibitory activity of Crn1 resides in its coiled coil domain. Localization of Crn1 to actin patches in vivo and association of Crn1 with the Arp2/3 complex also require its coiled coil domain. Genetic studies provide in vivo evidence for these interactions and activities. Overexpression of CRN1 causes growth arrest and redistribution of Arp2 and Crn1p into aberrant actin loops. These defects are suppressed by deletion of the Crn1 coiled coil domain and by arc35-26, an allele of the p35 subunit of the Arp2/3 complex. Further in vivo evidence that coronin regulates the Arp2/3 complex comes from the observation that crn1 and arp2 mutants display an allele-specific synthetic interaction. This work identifies a new form of regulation of the Arp2/3 complex and an important cellular function for coronin.

scholarly journals High-throughput analysis of candidate imprinted genes and allele-specific gene expression in the human term placenta

BMC Genetics ◽  
2010 ◽  
Vol 11 (1) ◽  
pp. 25 ◽  
Author(s):  
Caroline Daelemans ◽  
Matthew E Ritchie ◽  
Guillaume Smits ◽  
Sayeda Abu-Amero ◽  
Ian M Sudbery ◽  
...  
Keyword(s):  
Gene Expression ◽  
High Throughput ◽  
Specific Gene ◽  
Imprinted Genes ◽  
High Throughput Analysis ◽  
Human Term Placenta ◽  
Throughput Analysis ◽  
Term Placenta ◽  
Specific Gene Expression ◽  
Allele Specific

scholarly journals Improving the efficiency of precise genome editing with site-specific Cas9-oligonucleotide conjugates

2020 ◽  
Vol 6 (15) ◽  
pp. eaaz0051 ◽  
Author(s):  
Xinyu Ling ◽  
Bingteng Xie ◽  
Xiaoqin Gao ◽  
Liying Chang ◽  
Wei Zheng ◽  
...  
Keyword(s):  
Genome Editing ◽  
Therapeutic Potential ◽  
Chemical Reactivity ◽  
Specific Chemical ◽  
Site Specific ◽  
Additional Tool ◽  
Homology Directed Repair ◽  
Chemically Modified ◽  
Dna Template ◽  
Oligonucleotide Conjugates

Site-specific chemical conjugation of proteins can enhance their therapeutic and diagnostic utility but has seldom been applied to CRISPR-Cas9, which is a rapidly growing field with great therapeutic potential. The low efficiency of homology-directed repair remains a major hurdle in CRISPR-Cas9–mediated precise genome editing, which is limited by low concentration of donor DNA template at the cleavage site. In this study, we have developed methodology to site-specifically conjugate oligonucleotides to recombinant Cas9 protein containing a genetically encoded noncanonical amino acid with orthogonal chemical reactivity. The Cas9-oligonucleotide conjugates recruited an unmodified donor DNA template to the target site through base pairing, markedly increasing homology-directed repair efficiency in both human cell culture and mouse zygotes. These chemically modified Cas9 mutants provide an additional tool, one that is complementary to chemically modified nucleic acids, for improving the utility of CRISPR-Cas9–based genome-editing systems.

Concepts of Molecular Plant Breeding and Genome Editing

2021 ◽  
pp. 1-15
Keyword(s):  
Plant Breeding ◽  
Genome Editing ◽  
Genetic Linkage ◽  
Selection Process ◽  
Crop Improvement ◽  
Zinc Finger Nucleases ◽  
Induced Mutations ◽  
Transcription Activator ◽  
Targeted Gene Editing ◽  
Breeding Techniques

Traditional plant breeding depends on spontaneous and induced mutations available in the crop plants. Such mutations are rare and occur randomly. By contrast, molecular breeding and genome editing are advanced breeding techniques that can enhance the selection process and produce precisely targeted modifications in any crop. Identification of molecular markers, based on SSRs and SNPs, and the availability of high-throughput (HTP) genotyping platforms have accelerated the process of generating dense genetic linkage maps and thereby enhanced application of marker-assisted breeding for crop improvement. Advanced molecular biology techniques that facilitate precise, efficient, and targeted modifications at genomic loci are termed as “genome editing.” The genome editing tools include “zinc-finger nucleases (ZNFs),” “transcription activator-like effector nucleases (TALENs),” oligonucleotide-directed mutagenesis (ODM), and “clustered regularly interspersed short palindromic repeats (CRISPER/Cas) system,” which can be used for targeted gene editing. Concepts of molecular plant breeding and genome editing systems are presented in this chapter.

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