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Communications Biology Nov 2022Cas12a can process multiple sgRNAs from a single transcript of CRISPR array, conferring advantages in multiplexed base editing when incorporated into base editor...
Cas12a can process multiple sgRNAs from a single transcript of CRISPR array, conferring advantages in multiplexed base editing when incorporated into base editor systems, which is extremely helpful given that phenotypes commonly involve multiple genes or single-nucleotide variants. However, multiplexed base editing through Cas12a-derived base editors has been barely reported, mainly due to the compromised efficiencies and restricted protospacer-adjacent motif (PAM) of TTTV for wild-type Cas12a. Here, we develop Cas12a-mediated cytosine base editor (CBE) and adenine base editor (ABE) systems with elevated efficiencies and expanded targeting scope, by combining highly active deaminases with Lachnospiraceae bacterium Cas12a (LbCas12a) variants. We confirm that these CBEs and ABEs can perform efficient C-to-T and A-to-G conversions, respectively, on targets with PAMs of NTTN, TYCN, and TRTN. Notably, multiplexed base editing can be conducted using the developed CBEs and ABEs in somatic cells and embryos. These Cas12a variant-mediated base editors will serve as versatile tools for multiplexed point mutation, which is notably important in genetic improvement, disease modeling, and gene therapy.
Topics: Gene Editing; CRISPR-Cas Systems; Cytosine; Adenine; Point Mutation
PubMed: 36323848
DOI: 10.1038/s42003-022-04152-8 -
Bioinformatics (Oxford, England) Mar 2012The folding free energy is an important characteristic of proteins stability and is directly related to protein's wild-type function. The changes of protein's stability...
MOTIVATION
The folding free energy is an important characteristic of proteins stability and is directly related to protein's wild-type function. The changes of protein's stability due to naturally occurring mutations, missense mutations, are typically causing diseases. Single point mutations made in vitro are frequently used to assess the contribution of given amino acid to the stability of the protein. In both cases, it is desirable to predict the change of the folding free energy upon single point mutations in order to either provide insights of the molecular mechanism of the change or to design new experimental studies.
RESULTS
We report an approach that predicts the free energy change upon single point mutation by utilizing the 3D structure of the wild-type protein. It is based on variation of the molecular mechanics Generalized Born (MMGB) method, scaled with optimized parameters (sMMGB) and utilizing specific model of unfolded state. The corresponding mutations are built in silico and the predictions are tested against large dataset of 1109 mutations with experimentally measured changes of the folding free energy. Benchmarking resulted in root mean square deviation = 1.78 kcal/mol and slope of the linear regression fit between the experimental data and the calculations was 1.04. The sMMGB is compared with other leading methods of predicting folding free energy changes upon single mutations and results discussed with respect to various parameters.
AVAILABILITY
All the pdb files we used in this article can be downloaded from http://compbio.clemson.edu/downloadDir/mentaldisorders/sMMGB_pdb.rar.
SUPPLEMENTARY INFORMATION
Supplementary data are available at Bioinformatics online.
Topics: Humans; Mutation, Missense; Point Mutation; Protein Folding; Protein Stability; Proteins; Software; Thermodynamics
PubMed: 22238268
DOI: 10.1093/bioinformatics/bts005 -
Cell Apr 1999
Review
Topics: Animals; DNA; DNA Methylation; Models, Genetic; Point Mutation; RNA; Repetitive Sequences, Nucleic Acid
PubMed: 10219236
DOI: 10.1016/s0092-8674(00)80725-4 -
Scientific Reports Oct 2020The generation of a wide range of candidate antibodies is important for the successful development of drugs that simultaneously satisfy multiple requirements. To find...
The generation of a wide range of candidate antibodies is important for the successful development of drugs that simultaneously satisfy multiple requirements. To find cooperative mutations and increase the diversity of mutants, an in silico double-point mutation approach, in which 3D models of all possible double-point mutant/antigen complexes are constructed and evaluated using interaction analysis, was developed. Starting from an antibody with very high affinity, four double-point mutants were designed in silico. Two of the double-point mutants exhibited improved affinity or affinity comparable to that of the starting antibody. The successful identification of two active double-point mutants showed that a cooperative mutation could be found by utilizing information regarding the interactions. The individual single-point mutants of the two active double-point mutants showed decreased affinity or no expression. These results suggested that the two active double-point mutants cannot be obtained through the usual approach i.e. a combination of improved single-point mutants. In addition, a triple-point mutant, which combines the distantly located active double-point mutation and an active single-point mutation collaterally obtained in the process of the double-point mutation strategy, was designed. The triple-point mutant showed improved affinity. This finding suggested that the effects of distantly located mutations are independent and additive. The double-point mutation approach using the interaction analysis of 3D structures expands the design repertoire for mutants, and hopefully paves a way for the identification of cooperative multiple-point mutations.
Topics: Antibodies; Antigens; Models, Molecular; Mutation; Point Mutation; Thermodynamics; Thromboplastin
PubMed: 33067496
DOI: 10.1038/s41598-020-74545-4 -
Biomolecules Dec 2019Accurate prediction of protein stability changes resulting from amino acid substitutions is of utmost importance in medicine to better understand which mutations are...
Accurate prediction of protein stability changes resulting from amino acid substitutions is of utmost importance in medicine to better understand which mutations are deleterious, leading to diseases, and which are neutral. Since conducting wet lab experiments to get a better understanding of protein mutations is costly and time consuming, and because of huge number of possible mutations the need of computational methods that could accurately predict effects of amino acid mutations is of greatest importance. In this research, we present a robust methodology to predict the energy changes of a proteins upon mutations. The proposed prediction scheme is based on two step algorithm that is a Holdout Random Sampler followed by a neural network model for regression. The Holdout Random Sampler is utilized to analysis the energy change, the corresponding uncertainty, and to obtain a set of admissible energy changes, expressed as a cumulative distribution function. These values are further utilized to train a simple neural network model that can predict the energy changes. Results were blindly tested (validated) against experimental energy changes, giving Pearson correlation coefficients of 0.66 for Single Point Mutations and 0.77 for Multiple Point Mutations. These results confirm the successfulness of our method, since it outperforms majority of previous studies in this field.
Topics: Amino Acids; Databases, Protein; Machine Learning; Neural Networks, Computer; Point Mutation; Protein Stability; Proteins; Thermodynamics
PubMed: 31906171
DOI: 10.3390/biom10010067 -
Journal of Visualized Experiments : JoVE Aug 2017Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point mutations, which often...
Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point mutations, which often are responsible for a variety of human inherited disorders. Using a well-established cell-based model system, the point mutation of a single-copy mutant eGFP gene integrated into HCT116 cells has been repaired using this combinatorial approach. The analysis of corrected and uncorrected cells reveals both the precision of gene editing and the development of genetic lesions, when indels are created in uncorrected cells in the DNA sequence surrounding the target site. Here, the specific methodology used to analyze this combinatorial approach to the gene editing of a point mutation, coupled with a detailed experimental strategy to measuring indel formation at the target site, is outlined. This protocol outlines a foundational approach and workflow for investigations aimed at developing CRISPR/Cas9-based gene editing for human therapy. The conclusion of this work is that on-site mutagenesis takes place as a result of CRISPR/Cas9 activity during the process of point mutation repair. This work puts in place a standardized methodology to identify the degree of mutagenesis, which should be an important and critical aspect of any approach destined for clinical implementation.
Topics: CRISPR-Cas Systems; Cell Differentiation; Gene Editing; Humans; Mutagens; Mutation; Oligonucleotides; Point Mutation
PubMed: 28872131
DOI: 10.3791/56195 -
Frontiers of Medicine Jun 2014Activation-induced deaminase (AID) initiates the secondary antibody diversification process in B lymphocytes. In mammalian B cells, this process includes somatic... (Review)
Review
Activation-induced deaminase (AID) initiates the secondary antibody diversification process in B lymphocytes. In mammalian B cells, this process includes somatic hypermutation (SHM) and class switch recombination (CSR), both of which require AID. AID induces U:G mismatch lesions in DNA that are subsequently converted into point mutations or DNA double stranded breaks during SHM/CSR. In a physiological context, AID targets immunoglobulin (Ig) loci to mediate SHM/CSR. However, recent studies reveal genome-wide access of AID to numerous non-Ig loci. Thus, AID poses a threat to the genome of B cells if AID-initiated DNA lesions cannot be properly repaired. In this review, we focus on the molecular mechanisms that regulate the specificity of AID targeting and the repair pathways responsible for processing AID-initiated DNA lesions.
Topics: Animals; Antibody Diversity; B-Lymphocytes; Cytidine Deaminase; DNA Damage; DNA Repair; Immunoglobulin Class Switching; Point Mutation
PubMed: 24748462
DOI: 10.1007/s11684-014-0324-4 -
PLoS Computational Biology Nov 2015There is great interest in increasing proteins' stability to enhance their utility as biocatalysts, therapeutics, diagnostics and nanomaterials. Directed evolution is a...
There is great interest in increasing proteins' stability to enhance their utility as biocatalysts, therapeutics, diagnostics and nanomaterials. Directed evolution is a powerful, but experimentally strenuous approach. Computational methods offer attractive alternatives. However, due to the limited reliability of predictions and potentially antagonistic effects of substitutions, only single-point mutations are usually predicted in silico, experimentally verified and then recombined in multiple-point mutants. Thus, substantial screening is still required. Here we present FireProt, a robust computational strategy for predicting highly stable multiple-point mutants that combines energy- and evolution-based approaches with smart filtering to identify additive stabilizing mutations. FireProt's reliability and applicability was demonstrated by validating its predictions against 656 mutations from the ProTherm database. We demonstrate that thermostability of the model enzymes haloalkane dehalogenase DhaA and γ-hexachlorocyclohexane dehydrochlorinase LinA can be substantially increased (ΔTm = 24°C and 21°C) by constructing and characterizing only a handful of multiple-point mutants. FireProt can be applied to any protein for which a tertiary structure and homologous sequences are available, and will facilitate the rapid development of robust proteins for biomedical and biotechnological applications.
Topics: Computational Biology; Computer Simulation; Databases, Genetic; Enzyme Stability; Lyases; Models, Molecular; Point Mutation; Protein Engineering; Temperature
PubMed: 26529612
DOI: 10.1371/journal.pcbi.1004556 -
BMC Biotechnology Nov 2016Fission yeast is one of the most commonly used model organisms for studying genetics. For selection of desirable genotypes, antibiotic resistance cassettes are widely...
BACKGROUND
Fission yeast is one of the most commonly used model organisms for studying genetics. For selection of desirable genotypes, antibiotic resistance cassettes are widely integrated into the genome near genes of interest. In yeasts, this is achieved by PCR amplification of the cassette flanked by short homology sequences, which can be incorporated by homology directed repair. However, the currently available cassettes all share the same tef promoter and terminator sequences. It can therefore be challenging to perform multiple genetic modifications by PCR-based targeting, as existing resistance cassettes in strains can be favored for recombination due to shared homology between the cassettes.
RESULTS
Here we have generated new selection cassettes that do not recombine with those traditionally used. We achieved this by swapping the tef promoter and terminator sequences in the established antibiotic resistance MX6 cassette series for alternative promoters and/or terminators. The newly created selection cassettes did not recombine with the tef-containing MX6 cassettes already present in the genome, allowing for sequential gene targeting using the PCR-based method. In addition, we have generated a series of plasmids to facilitate the C-terminal tagging of genes with desired epitopes. We also utilized the anti-selection gene HSV-TK, which results in cell death in strains grown on the drug 5-Fluoro-2'-deoxyuridine (FdU, Floxuridin or FUDR). By fusing an antibiotic resistance gene to HSV-TK, we were able to select on the relevant antibiotic as well as counter-select on FdU media to confirm the desired genomic modification had been made. We noted that the efficiency of the counter selection by FdU was enhanced by treatment with hydroxyurea. However, a number of DNA replication checkpoint and homologous recombination mutants, including rad3∆, cds1∆, rad54∆ and rad55∆, exhibited sensitivity to FdU even though those strains did not carry the HSV-TK gene. To remove counter-selectable markers, we introduced the Cre-loxP irreversible recombination method. Finally, utilizing the negative selectable markers, we showed efficient induction of point mutations in an endogenous gene by a two-step transformation method.
CONCLUSIONS
The plasmid constructs and techniques described here are invaluable tools for sequential gene targeting and will simplify construction of fission yeast strains required for study.
Topics: DNA, Fungal; Drug Resistance, Fungal; Gene Targeting; Mutagenesis, Insertional; Point Mutation; Promoter Regions, Genetic; Schizosaccharomyces
PubMed: 27825338
DOI: 10.1186/s12896-016-0307-4 -
Autism Research : Official Journal of... Apr 2014Multiple candidate genes have been identified for autism spectrum disorders. While some of these genes reach genome-wide significance, others, such as the R451C point...
Multiple candidate genes have been identified for autism spectrum disorders. While some of these genes reach genome-wide significance, others, such as the R451C point mutation in the synaptic cell adhesion molecule neuroligin-3, appear to be rare. Interestingly, two brothers with the same R451C point mutation in neuroligin-3 present clinically on seemingly disparate sides of the autism spectrum. These clinical findings suggest genetic background may play a role in modifying the penetrance of a particular autism-associated mutation. Animal models may contribute additional support for such mutations as functionally relevant and can provide mechanistic insights. Previously, in collaboration with the Südhof laboratory, we reported that mice with an R451C substitution in neuroligin-3 displayed social deficits and enhanced spatial learning. While some of these behavioral abnormalities have since been replicated independently in the Südhof laboratory, observations from the Crawley laboratory failed to replicate these findings in a similar neuroligin-3 mutant mouse model and suggested that genetic background may contribute to variation in observations across laboratories. Therefore, we sought to replicate our findings in the neuroligin-3 R451C point mutant knock-in mouse model (NL3R451C) in a different genetic background. We backcrossed our NL3R451C mouse line onto a 129S2/SvPasCrl genetic background and repeated a subset of our previous behavioral testing. NL3R451C mice on a 129S2/SvPasCrl displayed social deficits, enhanced spatial learning, and increased locomotor activity. These data extend our previous findings that NL3R451C mice exhibit autism-relevant behavioral abnormalities and further suggest that different genetic backgrounds can modify this behavioral phenotype through epistatic genetic interactions.
Topics: Animals; Cell Adhesion Molecules, Neuronal; Child Development Disorders, Pervasive; DNA Mutational Analysis; Female; Genetic Association Studies; Male; Membrane Proteins; Mice; Mice, Knockout; Models, Genetic; Nerve Tissue Proteins; Point Mutation; Social Behavior; Spatial Learning
PubMed: 24619977
DOI: 10.1002/aur.1362