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Yi Chuan = Hereditas Nov 2022Prime editing is a newly developed CRISPR/Cas system-based genome editing technique. The effector of prime editor (PE) is termed PE2, which is generated by fusing a... (Review)
Review
Prime editing is a newly developed CRISPR/Cas system-based genome editing technique. The effector of prime editor (PE) is termed PE2, which is generated by fusing a reverse transcriptase (RT) with a Cas9 H840A nickase. The guide RNA of PE is termed prime editing guide RNA (pegRNA), which consists of a single guide RNA (sgRNA) with a 3' extension containing the RT template (RTT) and primer binding site (PBS). PE can install all 12 types of point mutations, small insertions and deletions and combinations thereof. Since its emergence in 2019, with the high versatility and specificity, PE has been applied to many living organisms, including animals, plants and bacteria. This led to many explorations of PE on gene therapy and genetic improvement in agriculture. In this review, we systematically describe the development, characteristics, optimizations, applications and security of PE. In addition, we discuss the future applications of PE. We expect that this review will help researchers to grasp and better use PE.
Topics: Animals; CRISPR-Cas Systems; Gene Editing; Plants; Point Mutation; RNA, Small Untranslated
PubMed: 36384993
DOI: 10.16288/j.yczz.22-156 -
Journal of Molecular Modeling Mar 2023Crimean-Congo haemorrhagic fever virus (CCHFV) has tripartite RNA genome and is endemic in various countries of Asia, Africa and Europe.
INTRODUCTION
Crimean-Congo haemorrhagic fever virus (CCHFV) has tripartite RNA genome and is endemic in various countries of Asia, Africa and Europe.
METHOD
The present study is focused on mutation profiling of CCHFV L segment and phylogenetic clustering of protein dataset into six CCHFV genotypes.
RESULTS
Phylogenetic tree rooted with NCBI reference sequence (YP_325663.1) indicated less divergence from genotype III and the sequences belonging to same genotypes have shown less divergence among each other. Mutation frequency at 729 mutated positions was calculated and 563, 49, 33, 46 and 38 amino acid positions were found to be mutated at mutation frequency intervals of 0-0.2, 0.21-0.4, 0.41-0.6, 0.61-0.8 and 0.81-1.0 respectively. Thirty-eight highly frequent mutations (0.81-1.0 interval) were found in all genotypes and mapping in L segment (encoded for RdRp) revealed four mutations (V2074I, I2134T/A, V2148A and Q2695H/R) in catalytic site domain and no mutation in OTU domain. Molecular dynamic simulation and in silico analysis showed that catalytic site domain displayed large deviation and fluctuation upon introduction of these point mutations.
CONCLUSION
Overall study provides strong evidence that OTU domain is highly conserved and less prone to mutation whereas point mutations recorded in catalytic domain have affected the stability of protein and were found to be persistent in the large population.
Topics: Catalytic Domain; Phylogeny; Mutation; Point Mutation; RNA
PubMed: 36877258
DOI: 10.1007/s00894-023-05487-7 -
Molecules (Basel, Switzerland) Aug 2021Cytochrome is a small globular protein whose main physiological role is to shuttle electrons within the mitochondrial electron transport chain. This protein has been... (Review)
Review
Cytochrome is a small globular protein whose main physiological role is to shuttle electrons within the mitochondrial electron transport chain. This protein has been widely investigated, especially as a paradigmatic system for understanding the fundamental aspects of biological electron transfer and protein folding. Nevertheless, cytochrome can also be endowed with a non-native catalytic activity and be immobilized on an electrode surface for the development of third generation biosensors. Here, an overview is offered of the most significant examples of such a functional transformation, carried out by either point mutation(s) or controlled unfolding. The latter can be induced chemically or upon protein immobilization on hydrophobic self-assembled monolayers. We critically discuss the potential held by these systems as core constituents of amperometric biosensors, along with the issues that need to be addressed to optimize their applicability and response.
Topics: Biosensing Techniques; Electrochemistry; Electrons; Oxidation-Reduction; Point Mutation; Protein Folding; Proteins
PubMed: 34443538
DOI: 10.3390/molecules26164950 -
Methods in Molecular Biology (Clifton,... 2021TALENs (Transcription Activator-Like EndoNuclease) are molecular scissors designed to recognize and introduce a double-strand break at a specific genome locus. They...
TALENs (Transcription Activator-Like EndoNuclease) are molecular scissors designed to recognize and introduce a double-strand break at a specific genome locus. They represent tools of interest in the frame of genome edition. Upon cleavage, two different pathways lead to DNA repair: Non-homologous End Joining (NHEJ) repair, leading to efficient introduction of short insertion/deletion mutations which can disrupt translational reading frame and Homology Recombination (HR)-directed repair that occurs when exogenous DNA is supplied. Here we introduce how to use TALENs in the oleaginous yeast Yarrowia lipolytica by presenting a step-by-step method allowing to knock out or to introduce in vivo a point mutation in a gene of Yarrowia lipolytica. This chapter describes the material required, the transformation procedure, and the screening process.
Topics: DNA End-Joining Repair; Gene Editing; Genome, Fungal; Point Mutation; Software; Transcription Activator-Like Effector Nucleases; Yarrowia
PubMed: 33847980
DOI: 10.1007/978-1-0716-1414-3_2 -
The Tohoku Journal of Experimental... Aug 2022Virus genome mutation profiles with insertion, deletion, and point mutations have recently been revealed to differ remarkably between viruses. In RNA viruses like human...
Virus genome mutation profiles with insertion, deletion, and point mutations have recently been revealed to differ remarkably between viruses. In RNA viruses like human coronaviruses or influenza viruses, genome samples collected over two to three decades usually show point mutations in 10-20% of the bases, while the rate of insertion and/or deletion mutations (indels) largely depends on the virus. This study evaluates the mutation profiles of DNA viruses by comparing a recently sampled genome of human adenovirus species C type 2 (isolate SG06/HAdvC2/2016) with a genome of the same species sampled in the 1970s. It was found insertions of 23 bases at seven sites and deletions of 22 bases at nine sites. The longest indels were 6-base insertions in E2B and L4. All indels in the coding regions were in-frame mutations with base lengths in multiples of three. In the non-coding regions, the lengths of the indels ranged from 1-4 consecutive bases. Long indels with more than 10 consecutive bases, which comprise nearly half of indels in the SARS-CoV-2 genome, were absent. In other sites, the point mutation rate was approximately 0.3%, which was significantly lower than in RNA viruses. In summary, the estimated point mutation rate in human adenovirus species C type 2 (HAdvC-2) was over 10 times lower than in RNA viruses. Unlike the relatively long indels in the SARS-CoV-2 genome, the indels in HAdvC-2 were short, with 6 or fewer consecutive bases.
Topics: Adenoviruses, Human; Genome, Viral; INDEL Mutation; Point Mutation; SARS-CoV-2
PubMed: 35705320
DOI: 10.1620/tjem.2022.J049 -
Cells Dec 2023We report the first correction from prime editing a mutation in the gene, paving the way to gene therapies for RYR1-related myopathies. The gene codes for a calcium...
We report the first correction from prime editing a mutation in the gene, paving the way to gene therapies for RYR1-related myopathies. The gene codes for a calcium channel named Ryanodine receptor 1, which is expressed in skeletal muscle fibers. The failure of this channel causes muscle weakness in patients, which leads to motor disabilities. Currently, there are no effective treatments for these diseases, which are mainly caused by point mutations. Prime editing allows for the modification of precise nucleotides in the DNA. Our results showed a 59% correction rate of the T4709M mutation in the gene in human myoblasts by RNA delivery of the prime editing components. It is to be noted that T4709M is recessive and, thus, persons having a heterozygous mutation are healthy. These results are the first demonstration that correcting mutations in the RYR1 gene is possible.
Topics: Humans; Muscle Weakness; Muscular Diseases; Mutation; Point Mutation; Ryanodine Receptor Calcium Release Channel
PubMed: 38201236
DOI: 10.3390/cells13010031 -
Frontiers in Immunology 2022Sphingosine-1-phosphate lyase is encoded by the gene, degrades S1P, and is crucial for S1P homeostasis in animal models and humans. S1P lyase deficient patients suffer...
Sphingosine-1-phosphate lyase is encoded by the gene, degrades S1P, and is crucial for S1P homeostasis in animal models and humans. S1P lyase deficient patients suffer from adrenal insufficiency, severe lymphopenia, and skin disorders. In this study, we used random mutagenesis screening to identify a mouse line carrying a missense mutation of (M467K). This mutation caused similar pathologies as Sgpl1 knock-out mice in multiple organs, but greatly preserved its lifespan, which M467K mutation mice look normal under SPF conditions for over 40 weeks, in contrast, the knock-out mice live no more than 6 weeks. When treated with Imiquimod, mice experienced exacerbated skin inflammation, as revealed by aggravated acanthosis and orthokeratotic hyperkeratosis. We also demonstrated that the IL17a producing Vγ6 cell was enriched in skin and caused severe pathology after imiquimod treatment. Interestingly, hyperchromic plaque occurred in the mutant mice one month after Imiquimod treatment but not in the controls, which resembled the skin disorder found in deficient patients. Therefore, our results demonstrate that point mutation mice successfully modeled a human disease after being treated with Imiquimod. We also revealed a major subset of γδT cells in the skin, IL17 secreting Vγ6 T cells were augmented by deficiency and led to skin pathology. Therefore, we have, for the first time, linked the IL17a and γδT cells to SPL insufficiency.
Topics: Animals; Homeostasis; Hyperpigmentation; Imiquimod; Mice; Mice, Knockout; Point Mutation
PubMed: 35769463
DOI: 10.3389/fimmu.2022.728455 -
Trends in Biotechnology Oct 2019Genome editing with CRISPR/Cas has rapidly gained popularity. Base editing, a new CRISPR/Cas-based approach, can precisely convert one nucleotide to another in DNA or... (Review)
Review
Genome editing with CRISPR/Cas has rapidly gained popularity. Base editing, a new CRISPR/Cas-based approach, can precisely convert one nucleotide to another in DNA or RNA without inducing a double-strand DNA break (DSB). A combination of catalytically impaired nuclease variants with different deaminases has yielded diverse base-editing platforms that aim to address the key limitations such as specificity, protospacer adjacent motif (PAM) compatibility, editing window length, bystander editing, and sequence context preference. Because new base editors significantly reduce unintended editing in the genome, they hold great promise for treating genetic diseases and for developing superior agricultural crops. We review here the development of various base editors, assess their technical advantages and limitations, and discuss their broad applications in basic research, medicine, and agriculture.
Topics: Adenine; Animals; Base Pairing; CRISPR-Cas Systems; Codon, Terminator; Cytosine; Gene Editing; Gene Expression; Genetic Therapy; Humans; INDEL Mutation; Point Mutation
PubMed: 30995964
DOI: 10.1016/j.tibtech.2019.03.008 -
Journal of the American Chemical Society Feb 2020Glycoside hydrolases and phosphorylases are two major classes of enzymes responsible for the cleavage of glycosidic bonds. Here we show that two GH84 -GlcNAcase enzymes...
Glycoside hydrolases and phosphorylases are two major classes of enzymes responsible for the cleavage of glycosidic bonds. Here we show that two GH84 -GlcNAcase enzymes can be converted to efficient phosphorylases by a single point mutation. Noteworthy, the mutated enzymes are over 10-fold more active than naturally occurring glucosaminide phosphorylases. We rationalize this novel transformation using molecular dynamics and QM/MM metadynamics methods, showing that the mutation changes the electrostatic potential at the active site and reduces the energy barrier for phosphorolysis by 10 kcal·mol. In addition, the simulations unambiguously reveal the nature of the intermediate as a glucose oxazolinium ion, clarifying the debate on the nature of such a reaction intermediate in glycoside hydrolases operating via substrate-assisted catalysis.
Topics: Catalytic Domain; Glycoside Hydrolases; Phosphorylases; Point Mutation
PubMed: 31917561
DOI: 10.1021/jacs.9b09655 -
G3 (Bethesda, Md.) Aug 2020The mutation rate and mutations' effects on fitness are crucial to evolution. Mutation rates are under selection due to linkage between mutation rate modifiers and...
The mutation rate and mutations' effects on fitness are crucial to evolution. Mutation rates are under selection due to linkage between mutation rate modifiers and mutations' effects on fitness. The linkage between a higher mutation rate and more beneficial mutations selects for higher mutation rates, while the linkage between a higher mutation rate and more deleterious mutations selects for lower mutation rates. The net direction of selection on mutations rates depends on the fitness landscape, and a great deal of work has elucidated the fitness landscapes of mutations. However, tests of the effect of varying a mutation rate on evolution in a single organism in a single environment have been difficult. This has been studied using strains of antimutators and mutators, but these strains may differ in additional ways and typically do not allow for continuous variation of the mutation rate. To help investigate the effects of the mutation rate on evolution, we have genetically engineered a strain of with a point mutation rate that can be smoothly varied over two orders of magnitude. We did this by engineering a strain with inducible control of the mismatch repair proteins MutH and MutL. We used this strain in an approximately 350 generation evolution experiment with controlled variation of the mutation rate. We confirmed the construct and the mutation rate were stable over this time. Sequencing evolved strains revealed a higher number of single nucleotide polymorphisms at higher mutations rates, likely due to either the beneficial effects of these mutations or their linkage to beneficial mutations.
Topics: Escherichia coli; Escherichia coli Proteins; Mutation; Mutation Rate; Point Mutation; Selection, Genetic
PubMed: 32503807
DOI: 10.1534/g3.120.401124