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BMC Genomics Jun 2023Rewriting the genomes of living organisms has been a long-standing aim in the biological sciences. The revelation of the CRISPR/Cas9 technology has revolutionized the...
BACKGROUND
Rewriting the genomes of living organisms has been a long-standing aim in the biological sciences. The revelation of the CRISPR/Cas9 technology has revolutionized the entire biological field. Since its emergence, this technology has been widely applied to induce gene knockouts, insertions, deletions, and base substitutions. However, the classical version of this system was imperfect for inducing or correcting desired mutations. A subsequent development generated more advanced classes, including cytosine and adenine base editors, which can be used to achieve single nucleotide substitutions. Nevertheless, these advanced systems still suffer from several limitations, such as the inability to edit loci without a suitable PAM sequence and to induce base transversions. On the other hand, the recently emerged prime editors (PEs) can achieve all possible single nucleotide substitutions as well as targeted insertions and deletions, which show promising potential to alter and correct the genomes of various organisms. Of note, the application of PE to edit livestock genomes has not been reported yet.
RESULTS
In this study, using PE, we successfully generated sheep with two agriculturally significant mutations, including the fecundity-related FecB p.Q249R and the tail length-related TBXT p.G112W. Additionally, we applied PE to generate porcine blastocysts with a biomedically relevant point mutation (KCNJ5 p.G151R) as a porcine model of human primary aldosteronism.
CONCLUSIONS
Our study demonstrates the potential of the PE system to edit the genomes of large animals for the induction of economically desired mutations and for modeling human diseases. Although prime-edited sheep and porcine blastocysts could be generated, the editing frequencies are still unsatisfactory, highlighting the need for optimizations in the PE system for efficient generation of large animals with customized traits.
Topics: Humans; Animals; Swine; Sheep; Mutation; Point Mutation; Blastocyst; Livestock; Nucleotides; G Protein-Coupled Inwardly-Rectifying Potassium Channels
PubMed: 37308830
DOI: 10.1186/s12864-023-09409-y -
Applied Microbiology and Biotechnology Feb 2024Metabolic engineering frequently makes use of point mutation and saturation mutation library creation. At present, sequencing is the only reliable and direct technique...
Metabolic engineering frequently makes use of point mutation and saturation mutation library creation. At present, sequencing is the only reliable and direct technique to detect point mutation and screen saturation mutation library. In this study, mismatch amplification mutation assay (MAMA) PCR was used to detect point mutation and screen saturation mutation library. In order to fine-tune the expression of odhA encoding 2-oxoglutarate dehydrogenase E1 component, a saturating mutant library of the RBS of odhA was created in Corynebacterium glutamicum P12 based on the CRISPR-Cas2a genome editing system, which increased the L-proline production by 81.3%. MAMA PCR was used to filter out 42% of the non-mutant transformants in the mutant library, which effectively reduced the workload of the subsequent fermentation test and the number of sequenced samples. The rapid and sensitive MAMA-PCR method established in this study provides a general strategy for detecting point mutations and improving the efficiency of mutation library screening. KEY POINTS: • MAMA PCR was optimized and developed to detect point mutation. • MAMA PCR greatly improves the screening efficiency of point mutation. • Attenuation of odhA expression in P12 effectively improves proline production.
Topics: Point Mutation; Mutation; Base Sequence; Corynebacterium glutamicum; Polymerase Chain Reaction
PubMed: 38305911
DOI: 10.1007/s00253-024-13036-2 -
Methods in Molecular Biology (Clifton,... 2024The zebrafish is an ideal model for functional analysis of genes at the molecular, protein, cell, organ, and organism levels. We have used zebrafish to analyze the...
The zebrafish is an ideal model for functional analysis of genes at the molecular, protein, cell, organ, and organism levels. We have used zebrafish to analyze the function of members of the protein tyrosine phosphatase (PTP) superfamily for more than two decades. The molecular genetic toolbox has significantly improved over the years. Currently, generating mutant lines that lack the function of a PTP gene is relatively straightforward by CRISPR/Cas9 technology-mediated generation of insertions or deletions in the target gene. In addition, generating point mutations using CRISPR/Cas9 technology and homology-directed repair (HDR) is feasible, albeit the success rate could be higher. Here, we describe the methods, including the tips and tricks, that we have used to generate knock-out and knock-in zebrafish lines in PTP genes successfully.
Topics: Animals; Zebrafish; Protein Tyrosine Phosphatases; Craniocerebral Trauma; Perciformes; Point Mutation
PubMed: 38147210
DOI: 10.1007/978-1-0716-3569-8_6 -
Physical Chemistry Chemical Physics :... Feb 2021Proton transfer along the hydrogen bonds of DNA can lead to the creation of short-lived, but biologically relevant point mutations that can further lead to gene mutation...
Proton transfer along the hydrogen bonds of DNA can lead to the creation of short-lived, but biologically relevant point mutations that can further lead to gene mutation and, potentially, cancer. In this work, the energy landscape of the canonical A-T and G-C base pairs (standard, amino-keto) to tautomeric A*-T* and G*-C* (non-standard, imino-enol) Watson-Crick DNA base pairs is modelled with density functional theory and machine-learning nudge-elastic band methods. We calculate the energy barriers and tunnelling rates of hydrogen transfer between and within each base monomer (A, T, G and C). We show that the role of tunnelling in A-T tautomerisation is statistically unlikely due to the presence of a small reverse reaction barrier. On the contrary, the thermal populations of the G*-C* point mutation could be non-trivial and propagate through the replisome. For the direct intramolecular transfer, the reaction is hindered by a substantial energy barrier. However, our calculations indicate that tautomeric bases in their monomeric form have remarkably long lifetimes.
Topics: Base Pairing; DNA; Density Functional Theory; Hydrogen Bonding; Isomerism; Models, Chemical; Point Mutation; Protons; Thermodynamics
PubMed: 33533770
DOI: 10.1039/d0cp05781a -
Journal of the American Association of... Aug 2022Mitochondrial disorders arise from DNA mutations in either the mitochondrial DNA (mtDNA) or nuclear DNA genomes. This article focuses on a mtDNA base-pair mutation...
Mitochondrial disorders arise from DNA mutations in either the mitochondrial DNA (mtDNA) or nuclear DNA genomes. This article focuses on a mtDNA base-pair mutation associated with neuropathy, ataxia, and retinitis pigmentosa and Leigh syndrome and the large-scale mtDNA deletion associated with Kearns-Sayre syndrome. Disease sequelae and management strategies are reviewed, along with implications for the nurse practitioner in primary or specialty care.
Topics: Humans; DNA, Mitochondrial; Point Mutation; Mitochondrial Diseases; Kearns-Sayre Syndrome; Mutation
PubMed: 36330549
DOI: 10.1097/JXX.0000000000000755 -
The CRISPR Journal Aug 2021
Topics: CRISPR-Cas Systems; Cytosine; Gene Editing; Guanidine; Humans; Mutagenesis; Point Mutation
PubMed: 34406044
DOI: 10.1089/crispr.2021.29134.kni -
BMC Biology Oct 2023Gene knockout and knock-in have been widely performed in large farm animals based on genome editing systems. However, many types of precise gene editing, including...
BACKGROUND
Gene knockout and knock-in have been widely performed in large farm animals based on genome editing systems. However, many types of precise gene editing, including targeted deletion, gene tagging, and large gene fragment replacement, remain a challenge in large farm animals.
RESULTS
Here, we established versatile self-excising gene-targeting technology in combination with programmable nucleases (SEGCPN) to efficiently generate various types of precise gene editing in bovine. First, we used this versatile method to successfully generate bovine embryos with point mutations and 11-bp deletions at the MSTN locus. Second, we successfully generated bulls with EGFP labeling at the SRY locus. Finally, we successfully generated humanized cows in which the endogenous 18-kb α-casein gene was replaced with a 2.6-kb human α-lactalbumin gene.
CONCLUSIONS
In summary, our new SEGCPN method offers unlimited possibilities for various types of precise gene editing in large animals for application both in agriculture and disease models.
Topics: Female; Animals; Cattle; Male; Humans; Gene Editing; CRISPR-Cas Systems; Gene Targeting; Gene Knockout Techniques; Point Mutation
PubMed: 37864194
DOI: 10.1186/s12915-023-01677-0 -
Genes Apr 2023Cytosine base editors (CBEs) and adenine base editors (ABEs) are recently developed CRISPR-mediated genome-editing tools that do not introduce double-strand breaks. In...
Cytosine base editors (CBEs) and adenine base editors (ABEs) are recently developed CRISPR-mediated genome-editing tools that do not introduce double-strand breaks. In this study, five ABEs, ABE7.10, ABEmax, NG-ABEmax, ABE8e and NG-ABE8e, were used to generate A-to-G (T-to-C) conversions in five genome loci in porcine fetal fibroblasts (PFFs). Variable yet appreciable editing efficiencies and variable activity windows were observed in these targeting regions via these five editors. The strategy of two sgRNAs in one vector exhibited superior editing efficiency to that of using two separate sgRNA expression vectors. ABE-mediated start-codon mutation in silenced its expression of protein and, unexpectedly, eliminated the vast majority of its mRNA. No off-target DNA site was detected for these editors. Substantial off-target RNA events were present in the ABE-edited cells, but no KEGG pathway was found to be significantly enriched. Our study supports that ABEs are powerful tools for A-to-G (T-to-C) point-mutation modification in porcine cells.
Topics: Animals; Swine; Adenine; Gene Editing; Mutation; Point Mutation; Fibroblasts
PubMed: 37107666
DOI: 10.3390/genes14040908 -
BMC Bioinformatics Oct 2020Genomic profiling of solid human tumors by projects such as The Cancer Genome Atlas (TCGA) has provided important information regarding the somatic alterations that...
BACKGROUND
Genomic profiling of solid human tumors by projects such as The Cancer Genome Atlas (TCGA) has provided important information regarding the somatic alterations that drive cancer progression and patient survival. Although researchers have successfully leveraged TCGA data to build prognostic models, most efforts have focused on specific cancer types and a targeted set of gene-level predictors. Less is known about the prognostic ability of pathway-level variables in a pan-cancer setting. To address these limitations, we systematically evaluated and compared the prognostic ability of somatic point mutation (SPM) and copy number variation (CNV) data, gene-level and pathway-level models for a diverse set of TCGA cancer types and predictive modeling approaches.
RESULTS
We evaluated gene-level and pathway-level penalized Cox proportional hazards models using SPM and CNV data for 29 different TCGA cohorts. We measured predictive accuracy as the concordance index for predicting survival outcomes. Our comprehensive analysis suggests that the use of pathway-level predictors did not offer superior predictive power relative to gene-level models for all cancer types but had the advantages of robustness and parsimony. We identified a set of cohorts for which somatic alterations could not predict prognosis, and a unique cohort LGG, for which SPM data was more predictive than CNV data and the predictive accuracy is good for all model types. We found that the pathway-level predictors provide superior interpretative value and that there is often a serious collinearity issue for the gene-level models while pathway-level models avoided this issue.
CONCLUSION
Our comprehensive analysis suggests that when using somatic alterations data for cancer prognosis prediction, pathway-level models are more interpretable, stable and parsimonious compared to gene-level models. Pathway-level models also avoid the issue of collinearity, which can be serious for gene-level somatic alterations. The prognostic power of somatic alterations is highly variable across different cancer types and we have identified a set of cohorts for which somatic alterations could not predict prognosis. In general, CNV data predicts prognosis better than SPM data with the exception of the LGG cohort.
Topics: DNA Copy Number Variations; Humans; Point Mutation; Prognosis
PubMed: 33081688
DOI: 10.1186/s12859-020-03791-0 -
BMC Medical Genomics Jan 2021In addition to ovarian and breast cancers, loss-of-function mutations in BRCA1 and BRCA2 genes are also linked to an increased risk of pancreatic cancer, with ~ 4 to...
BACKGROUND
In addition to ovarian and breast cancers, loss-of-function mutations in BRCA1 and BRCA2 genes are also linked to an increased risk of pancreatic cancer, with ~ 4 to 7% of pancreatic cancer patients harboring germline BRCA mutations. Most BRCA alterations in pancreatic cancer are frame-shifting indels, stop-gain, and splice-site mutations, but single nucleotide substitutions are rare. Recent studies demonstrated a significant progression-free survival (PFS) benefit from maintenance olaparib, a poly (ADP-ribose) polymerase (PARP) inhibitor administered to patients with germline BRCA mutations and metastatic pancreatic cancer.
CASE PRESENTATION
Here, we report a metastatic pancreatic cancer case who harbored a novel somatic BRCA2 c.6944T > C (p. I2315T) point mutation. After 6 weeks first-line chemotherapy, the patient was refractory to treatment and had a progressive disease. Due to the novel nonsynonymous BRCA2 point mutation, we decided to change the strategy by administering olaparib. The patient benefited from olaparib therapy and achieved a PFS of ~ 6.5 months.
CONCLUSIONS
We describe a patient carrying a novel somatic BRCA2 p. I2315T point mutation, which is first reported in metastatic pancreatic cancer. This case report indicates that a gene mutation-based strategy should be considered in the clinic to provide more effective treatment.
Topics: Female; Genes, BRCA2; Humans; Pancreatic Neoplasms; Phthalazines; Piperazines; Point Mutation
PubMed: 33407459
DOI: 10.1186/s12920-020-00850-6