Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Gene Editing”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 487 records · Page 27Linked to original sources
PubMed2025

SelectRepair Knockout: Efficient PTC-Free Gene Knockout Through Selectable Homology-Directed DNA Repair.

Generating nonessential gene knockouts using CRISPR/Cas9 technology is becoming increasingly common in biological research. In a typical workflow, the Cas9 endonuclease is used to induce a DNA double-strand break that relies on nonhomologous end-joining (NHEJ) to introduce a premature termination codon (PTC) in the target gene. The goal is to isolate clones in which the gene produces PTC-containing mRNA transcripts that are degraded via nonsense-mediated mRNA decay (NMD) to cause loss of gene function. Unfortunately, this approach is laborious, and not all PTCs trigger NMD. More importantly, mounting evidence suggest that PTC mutations can also result in a transcriptional adaptation response that can mask the effects of a PTC-mediated gene knockout. In this chapter, we present a PTC-free gene knockout strategy that implements homology-directed DNA repair (HDR) with selectable markers to substantially reduce the complexity of the screening and validation of genome edits in cells containing more than one gene copy as in the case of the commonly used hypotriploid HEK293 cell line. We describe how to obtain a complete knockout of the Ligase IV protein (LIG4) and provide considerations for the application of this SelectRepair Knockout method to other genes.

Humans↗
PubMed2000

Gene dose-dependent maturation and receptor editing of B cells expressing immunoglobulin (Ig)G1 or IgM/IgG1 tail antigen receptors.

Conserved differences between the transmembrane and cytoplasmic domains of membrane immunoglobulin (Ig)M and IgG may alter the function of antigen receptors on naive versus memory B cells. Here, we compare the ability of these domains to signal B cell allelic exclusion and maturation in transgenic mice. A lysozyme-binding antibody was expressed in parallel sets of mice as IgM, IgG1, or a chimeric receptor with IgM extracellular domains and transmembrane/cytoplasmic domains of IgG1. Like IgM, the IgG1 or chimeric IgM/G receptors triggered heavy chain allelic exclusion and supported development of mature CD21(+) B cells. Many of the IgG or IgM/G B cells became CD21(high) and downregulated their IgG and IgM/G receptors spontaneously, resembling memory B cells and B cells with mutations that exaggerate B cell antigen receptor signaling. Unlike IgM-transgenic mice, "edited" B cells that carry non-hen egg lysozyme binding receptors preferentially accumulated in IgG and IgM/G mice. This was most extreme in lines with the highest transgene copy number and diminished in variant offspring with fewer copies. The sensitivity of B cell maturation to transgene copy number conferred by the IgG transmembrane and cytoplasmic domains may explain the diverse phenotypes found in other IgG-transgenic mouse strains and may reflect exaggerated signaling.

Animals↗
PubMed2002

Haplotype exclusion and receptor editing: irreconcilable differences?

Features of antibody genes and their regulation hinder two properties thought to be critical for clonal selection: haplotype exclusion and receptor diversity. These properties include: (1) the retention of multiple independent L-chain isotypes, which compounds the problem of allelic exclusion with one of isotype exclusion; (2) the process of receptor editing, in which recombination continues in cells already expressing antigen receptors; and (3) non-random associations and quasi-ordered rearrangements of the elements that generate light chain genes, which promote editing at the expense of allelic exclusion and receptor diversification. In contrast, heavy chain gene structure seems to promote haplotype exclusion and receptor diversity. It appears that requirements of receptor selection, such as the need for receptor editing as an immune tolerance mechanism and positive selection as a quality control checkpoint for receptor functionality, impose independent selections that shape the organization and regulation of the antibody genes. Despite these features, B cell development still achieves a significant level of phenotypic haplotype exclusion, suggesting that there is indeed significant selection for antibody monospecificity that is accommodated along with receptor editing. Thus, the immune system achieves both receptor selection and clonal selection, despite their partly antagonistic mechanisms.

Alleles↗
PubMed2001

The genomics of land plant chloroplasts: Gene content and alteration of genomic information by RNA editing.

The entire nucleotide sequence of the chloroplast genome has been determined from 12 land plants. The gene content and arrangement are relatively uniform from species to species, and the genome contains an average of 111 identified gene species (except Epifagus). Chloroplast genes can be classified into three main categories: Genes for the photosynthetic apparatus, those for the transcription/translation system, and those related to biosyntheses. The genes encoding components of the photosynthesis apparatus have been identified by protein chemical analyses from higher plants, Chlamydomonas and cyanobacteria, and then by chloroplast transformation techniques using tobacco and Chlamydomonas. The genes for subunits of RNA polymerases and of ribosomes were initially deduced similarity to those in E. coli, and later confirmed by protein analyses. Coding information is often modified at the level of transcripts by RNA editing (mostly C-U changes), resulting in amino acid substitutions and creation of novel reading frames. Perspectives of chloroplast genomics are discussed.

Journal Article↗
PubMed1995

Complete sequence and gene organization of the mitochondrial genome of the land snail Albinaria coerulea.

The complete sequence (14,130 bp) of the mitochondrial DNA (mtDNA) of the land snail Albinaria coerulea was determined. It contains 13 protein, two rRNA and 22 tRNA genes. Twenty-four of these genes are encoded by one and 13 genes by the other strand. The gene arrangement shares almost no similarities with that of two other molluscs for which the complete gene content and arrangement are known, the bivalve Mytilus edulis and the chiton Katharina tunicata; the protein and rRNA gene order is similar to that of another terrestrial gastropod, Cepaea nemoralis. Unusual features include the following: (1) the absence of lengthy noncoding regions (there are only 141 intergenic nucleotides interspersed at different gene borders, the longest intergenic sequence being 42 nucleotides) (2) the presence of several overlapping genes (mostly tRNAs), (3) the presence of tRNA-like structures and other stem and loop structures within genes. An RNA editing system acting on tRNAs must necessarily be invoked for posttranscriptional extension of the overlapping tRNAs. Due to these features, and also because of the small size of its genes (e.g., it contains the smallest rRNA genes among the known coelomates), it is one of the most compact mitochondrial genomes known to date.

Animals↗
PubMed1994

Effect of fat feeding on human intestinal apolipoprotein B mRNA levels and editing.

Our purpose was to assess the effect of a fat-rich meal on intestinal apolipoprotein B (apoB) mRNA levels and editing. We obtained jejunal biopsies from eight healthy adults in the fasting state and 3 h after a meal containing 1 g/kg of fat. In the fasting state, 93% of the apoB mRNA contained the editing sequence resulting in apoB-48 production. Feeding induced no significant changes in apoB mRNA levels or editing. Our data are consistent with the concept that the significant increase in apoB-48 within triglyceride-rich lipoproteins in postprandial plasma is not due to alterations in apoB gene expression or editing.

Apolipoproteins B↗
PubMed2002

Micro-processing events in mRNAs identified by DHPLC analysis.

Post-transcriptional processes such as alternative splicing and RNA editing have a huge impact on the diversity of the proteome. Detecting alternatively spliced transcripts is difficult when they are rare. In addition, edited transcripts often differ from the genomic sequence by only a few nucleotides. Denaturing high performance liquid chromatography (DHPLC) is routinely used for single nucleotide polymorphism detection and we used this method to detect alternatively spliced or edited transcripts. As the sites of RNA editing appear to be conserved within gene families, we investigated whether editing sites are conserved in the murine homologue of the Drosophila cacophony transcript encoding the alpha1 subunit of a voltage-gated calcium channel that is edited at 10 independent positions. Although DHPLC analysis detects RNA editing at as low as 3% in control transcripts, no evidence of RNA editing was found in the analysed murine transcript. However an alternative exon was identified at the 3' end of the mouse Cacna1alpha transcript and an alternative micro-exon encoding only two amino acids (NP) was found in the extracellular loop before the IVS4 helix in the same transcript. In the homologous Drosophila transcript a micro-exon also encoding two amino acids was found at the same position before the IVS4 helix.

Alternative Splicing↗
PubMed2005

Model for codon position bias in RNA editing.

RNA editing can be crucial for the expression of genetic information via inserting, deleting, or substituting a few nucleotides at specific positions in an RNA sequence. Within coding regions in an RNA sequence, editing usually occurs with a certain bias in choosing the positions of the editing sites. In the mitochondrial genes of Physarum polycephalum, many more editing events have been observed at the third codon position than at the first and second, while in some plant mitochondria the second codon position dominates. Here we propose an evolutionary model that explains this bias as the basis of selection at the protein level. The model predicts a distribution of the three positions rather close to the experimental observation in Physarum. This suggests that the codon position bias in Physarum is mainly a consequence of selection at the protein level.

Animals↗
PubMed2024

Lung and liver editing by lipid nanoparticle delivery of a stable CRISPR-Cas9 ribonucleoprotein.

Lipid nanoparticle (LNP) delivery of clustered regularly interspaced short palindromic repeat (CRISPR) ribonucleoproteins (RNPs) could enable high-efficiency, low-toxicity and scalable in vivo genome editing if efficacious RNP-LNP complexes can be reliably produced. Here we engineer a thermostable Cas9 from Geobacillus stearothermophilus (GeoCas9) to generate iGeoCas9 variants capable of >100× more genome editing of cells and organs compared with the native GeoCas9 enzyme. Furthermore, iGeoCas9 RNP-LNP complexes edit a variety of cell types and induce homology-directed repair in cells receiving codelivered single-stranded DNA templates. Using tissue-selective LNP formulations, we observe genome-editing levels of 16‒37% in the liver and lungs of reporter mice that receive single intravenous injections of iGeoCas9 RNP-LNPs. In addition, iGeoCas9 RNPs complexed to biodegradable LNPs edit the disease-causing SFTPC gene in lung tissue with 19% average efficiency, representing a major improvement over genome-editing levels observed previously using viral or nonviral delivery strategies. These results show that thermostable Cas9 RNP-LNP complexes can expand the therapeutic potential of genome editing.

Gene Editing↗
PubMed2002

Inhibition of hepatitis delta virus RNA editing by short inhibitory RNA-mediated knockdown of ADAR1 but not ADAR2 expression.

Hepatitis delta virus (HDV) requires host RNA editing at the viral RNA amber/W site. Of the two host genes responsible for RNA editing via deamination of adenosines in double-stranded RNAs, short inhibitory RNA-mediated knockdown of host ADAR1 expression but not that of ADAR2 led to decreased HDV amber/W editing and virus production. Despite substantial sequence and structural variation among the amber/W sites of the three HDV genotypes, ADAR1a was primarily responsible for editing all three. We conclude that ADAR1 is primarily responsible for editing HDV RNA at the amber/W site during HDV infection.

Adenosine Deaminase↗
PubMed2005

The double-RNA-specific adenosine deaminase (DSRAD) gene in dyschromatosis symmetrica hereditaria patients: two novel mutations and one previously described.

BACKGROUND: Dyschromatosis symmetrica hereditaria (DSH, MIM 127400) is an autosomal dominant pigmentary genodermatosis. Pathogenic mutations in the double-RNA-specific adenosine deaminase (DSRAD) gene encoding an RNA editing enzyme have recently been identified. OBJECTIVES: To identify gene mutations of DSRAD in Chinese patients with DSH. METHODS: Three unrelated Chinese patients with DSH were subjected to mutation detection in DSRAD. Two had family histories of DSH. All the coding exons and their flanking sequences were amplified and sequenced. RESULTS: All three patients had heterozygous mutations including one non-sense, one frameshift and one missense mutation in DSRAD. CONCLUSIONS: Two novel mutations, c.3169delC (p.L1057fs) and c.3247C-->T (p.R1083C), and one recurrent mutation c.1420C-->T (p.R474X), were found in this series of Chinese patients with DSH.

Adenosine Deaminase↗
PubMed1999

Knockout of the glutamate dehydrogenase gene in bloodstream Trypanosoma brucei in culture has no effect on editing of mitochondrial mRNAs.

Glutamate dehydrogenase (GDH) was shown previously to bind the 3' oligo[U] tail of the mitochondrial guide RNAs (gRNAs) of Leishmania tarentolae, apparently in the dinucleotide pocket (Bringaud F, Stripecke R, Frech GC, Freedland S, Turck C, Byrne EM, Simpson L. Mol. Cell. Biol. 1997; 17:3915-3923). Bloodstream Trypanosoma brucei cells in culture represent a good system to investigate the genetic effects of knocking out kinetoplastid nuclear genes to test a role in RNA editing, since editing of several mitochondrial genes occurs but is dispensable for viability (Corell RA, Myler P, Stuart K. Mol. Biochem. Parasitol. 1994; 64:65-74 and Stuart K. In: Benne R, editor. RNA editing--the alteration of protein coding sequences of RNA. New York: Ellis Horwood, 1993:25-52). Both GDH alleles of bloodstream T. brucei in culture were replaced by drug resistant markers without any effect on viability. The ratios of edited to unedited mRNAs for several cryptogenes were assayed by primer extension analysis. The steady state abundances of these edited RNAs were unaffected by the double knockout. This evidence suggests that GDH may not play a role in the editing reaction in bloodstream trypanosomes in culture, but this conclusion is tentative since there could be redundant genes for any biological function. We employed a double allelic replacement technique to generate a tetracycline inducible conditional expression of an ectopic copy of the deleted gene in bloodstream trypanosomes in culture. We used this strategy for genes encoding mitochondrial proteins which are not required during this stage of the life cycle, but as a general strategy it should be appropriate for generation of conditional null mutants for essential genes as well.

Amino Acid Sequence↗
PubMed2026

Molecular Bases and Genetic Design of Rice Disease Resistance for Optimized Yield and Sustainable Agriculture.

Rice diseases continue to undermine yield stability and threaten the sustainability of rice production. The central challenge is therefore not simply to maximize immune activation, but to identify genetic interventions that remain effective across diverse pathogen races and environmental conditions without imposing excessive penalties on growth or yield. Here, we synthesize the molecular basis of rice immunity from a design-oriented perspective. We first examine cell-surface pattern-recognition receptors and intracellular nucleotide-binding leucine-rich repeat receptors, and then assess the shared signaling hubs and defence outputs that connect pathogen perception to antimicrobial responses. Rather than treating these components as equivalent breeding targets, we compare their translational potential according to resistance spectrum, anticipated durability, tunability, pleiotropic risk, and the strength of field evidence. We further discuss breeding strategies based on receptor engineering, editing of susceptibility genes and cis-regulatory elements, post-translational motif engineering, pathogen-inducible and upstream open reading frame-mediated regulation, resistance-gene stacking and artificial intelligence-assisted prediction. We argue that rational resistance design in rice should move beyond constitutive immune activation toward allele-specific, quantitative, spatially restricted and infection-responsive regulation. Integrating mechanistic insights with precision genome editing, accelerated breeding and responsible deployment offers a practical route to durable, yield-compatible disease resistance while reducing dependence on chemical control.

breeding strategy↗
PubMed1989

Structural and dynamic differences between normal and transforming N-ras gene products: a 31P and isotope-edited 1H NMR study.

[15N]Glycine was biosynthetically incorporated into normal cellular N-ras p21 and a position 12 transforming mutant, in order to produce p21 proteins containing several site-specific NMR probes at or near activating positions in the guanine nucleotide binding domain. We have previously assigned all five glycine resonances located in loops directly involved in binding of guanosine diphosphate in the wild-type p21 protein [Campbell-Burk, S., Papastavros, M. Z., McCormick, F., & Redfield, A. G. (1989) Proc. Natl. Acad. Sci. U.S.A 86, 817-820]. In this report, the corresponding glycine resonances in the p21 mutant have been assigned, and spectral differences between normal and mutant p21-guanosine diphosphate (p21.GDP) complexes have been investigated. Our combined 1H[15N] and 31P NMR results show that substitution of aspartate for glycine-12 produces perturbations in the phosphoryl binding domain, near the point of the mutation. Although many of the remaining glycines were unaffected, spectral differences were also observed outside the GDP binding domain. Two of the five active-site glycines in wild-type p21.GDP have very slow amide proton exchange rates with water (kappa less than 2.8 x 10(-5) s-1). The active-site glycines are located in solvent-exposed loops, so their apparent solvent inaccessibility may result from strong hydrogen bond formation between glycine amide protons and bound guanine diphosphate and/or other nearby groups in p21.

Aspartic Acid↗
PubMed1999

The versatility of paramyxovirus RNA polymerase stuttering.

Paramyxoviruses cotranscriptionally edit their P gene mRNAs by expanding the number of Gs of a conserved AnGn run. Different viruses insert different distributions of guanylates, e.g., Sendai virus inserts a single G, whereas parainfluenza virus type 3 inserts one to six Gs. The sequences conserved at the editing site, as well as the experimental evidence, suggest that the insertions occur by a stuttering process, i.e., by pseudotemplated transcription. The number of times the polymerase "stutters" at the editing site before continuing strictly templated elongation is directed by a cis-acting sequence found upstream of the insertions. We have examined the stuttering process during natural virus infections by constructing recombinant Sendai viruses with mutations in their cis-acting sequences. We found that the template stutter site is precisely determined (C1052) and that a relatively short region (approximately 6 nucleotides) just upstream of the AnGn run can modulate the overall frequency of mRNA editing as well as the distribution of the nucleotide insertions. The positions more proximal to the 5' AnGn run are the most important in this respect. We also provide evidence that the stability of the mRNA/template hybrid plays a determining role in the overall frequency and range of mRNA editing. When the template U run is extended all the way to the stutter site, adenylates rather than guanylates are added at the editing site and their distribution begins to resemble the polyadenylation associated with mRNA 3' end formation by the viral polymerase. Our data suggest how paramyxovirus mRNA editing and polyadenylation are related mechanistically and how editing sites may have evolved from poly(A)-termination sites or vice versa.

5' Untranslated Regions↗
PubMed1994

Rps3 and rpl16 genes do not overlap in Oenothera mitochondria: GTG as a potential translation initiation codon in plant mitochondria?

Characterization of the Oenothera mitochondrial ribosomal gene cluster rps19-rps3-rpl16 shows the two genes rps3 and rpl16 to be separated by 9 nucleotides. The first codon of rpl16 is a GTG codon for valine and the only potential translational start. This GTG codon is conserved at the same position in maize, Petunia and Marchantia mitochondria, while sequences diverge upstream. These observations suggest that GTG at least at this position may act as translation initiation codon in plant mitochondria. Analysis of RNA editing suggests both genes to code for functional ribosomal proteins in Oenothera mitochondria. A duplication/recombination event at a decanucleotide in the intron of rps3 created a pseudogene missing part of the intron and the 3' exon.

Amino Acid Sequence↗
PubMed2000

Novel gene conversion between X-Y homologues located in the nonrecombining region of the Y chromosome in Felidae (Mammalia).

Genes located on the mammalian Y chromosome outside of the pseudoautosomal region do not recombine with those on the X and are predicted to either undergo selection for male function or gradually degenerate because of an accumulation of deleterious mutations. Here, phylogenetic analyses of X-Y homologues, Zfx and Zfy, among 26 felid species indicate two ancestral episodes of directed genetic exchange (ectopic gene conversion) from X to Y: once during the evolution of pallas cat and once in a common predecessor of ocelot lineage species. Replacement of the more rapidly evolving Y homologue with the evolutionarily constrained X copy may represent a mechanism for adaptive editing of functional genes on the nonrecombining region of the mammalian Y chromosome.

Animals↗