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Disease Models & Mechanisms Oct 2018The zebrafish is an increasingly popular model organism for human genetic disease research. CRISPR/Cas9-based approaches are currently used for multiple gene-editing...
The zebrafish is an increasingly popular model organism for human genetic disease research. CRISPR/Cas9-based approaches are currently used for multiple gene-editing purposes in zebrafish, but few studies have developed reliable ways to introduce precise mutations. Point mutation knock-in using CRISPR/Cas9 and single-stranded oligodeoxynucleotides (ssODNs) is currently the most promising technology for this purpose. Despite some progress in applying this technique to zebrafish, there is still a great need for improvements in terms of its efficiency, optimal design of sgRNA and ssODNs and broader applicability. The papers discussed in this Editorial provide excellent case studies on identifying problems inherent in the mutation knock-in technique, quantifying these issues and proposing strategies to overcome them. These reports also illustrate how the procedures for introducing specific mutations can be straightforward, such that ssODNs with only the target mutation are sufficient for generating the intended knock-in animals. Two of the studies also develop interesting point mutant knock-in models for cardiac diseases, validating the translational relevance of generating knock-in mutations and opening the door to many possibilities for their further study.
Topics: Animals; CRISPR-Cas Systems; Disease Models, Animal; Gene Knock-In Techniques; Point Mutation; Zebrafish
PubMed: 30366936
DOI: 10.1242/dmm.037515 -
Nature Communications Sep 2023Cas12a, a CRISPR-associated protein complex, has an inherent ability to cleave DNA substrates and is utilized in diagnostic tools to identify DNA molecules. We...
Cas12a, a CRISPR-associated protein complex, has an inherent ability to cleave DNA substrates and is utilized in diagnostic tools to identify DNA molecules. We demonstrate that multiple orthologs of Cas12a activate trans-cleavage in the presence of split activators. Specifically, the PAM-distal region of the crRNA recognizes RNA targets provided that the PAM-proximal seed region has a DNA target. Our method, Split Activator for Highly Accessible RNA Analysis (SAHARA), detects picomolar concentrations of RNA without sample amplification, reverse-transcription, or strand-displacement by simply supplying a short DNA sequence complementary to the seed region. Beyond RNA detection, SAHARA outperforms wild-type CRISPR-Cas12a in specificity towards point-mutations and can detect multiple RNA and DNA targets in pooled crRNA/Cas12a arrays via distinct PAM-proximal seed DNAs. In conclusion, SAHARA is a simple, yet powerful nucleic acid detection platform based on Cas12a that can be applied in a multiplexed fashion and potentially be expanded to other CRISPR-Cas enzymes.
Topics: RNA; CRISPR-Cas Systems; Point Mutation; RNA, Guide, CRISPR-Cas Systems; Recombination, Genetic
PubMed: 37669948
DOI: 10.1038/s41467-023-41006-1 -
Current Opinion in Structural Biology Jun 2011The evolution of disordered proteins or regions of proteins differs from that of ordered proteins because of the differences in their sequence composition,... (Review)
Review
The evolution of disordered proteins or regions of proteins differs from that of ordered proteins because of the differences in their sequence composition, intramolecular contacts, and function. Recent assessments of disordered protein evolution at the sequence, structural, and functional levels support this hypothesis. Disordered proteins have a different pattern of accepted point mutations, exhibit higher rates of insertions and deletions, and generally, but not always, evolve more rapidly than ordered proteins. Even with these high rates of sequence evolution, a few examples have shown that disordered proteins maintain their flexibility under physiological conditions, and it is hypothesized that they maintain specific structural ensembles.
Topics: Animals; Catalytic Domain; Evolution, Molecular; Humans; Point Mutation; Protein Binding; Protein Conformation; Protein Folding; Proteins
PubMed: 21482101
DOI: 10.1016/j.sbi.2011.02.005 -
Zhejiang Da Xue Xue Bao. Yi Xue Ban =... Apr 2021To establish a rabbit model of proprotein convertase subtilisin/kexin type9 () point mutation with CRISPR/Cas9 gene editing technique. According to the PubMed gene...
To establish a rabbit model of proprotein convertase subtilisin/kexin type9 () point mutation with CRISPR/Cas9 gene editing technique. According to the PubMed gene protein data, the PCSK9 protein functional regions of human and rabbit were analyzed by Blast. The 386S (Ser) amino acid functional region of human gene was homologous to the 485S of rabbit gene. Three small guide RNAs and one single-stranded donor oligonucleotide were designed according to the 485S base substitution position and sequence analysis of rabbit gene. The synthetic small guide RNAs, Cas9 mRNA and single-stranded donor oligonucleotide were co-injected into the cytoplasm of rabbit fertilized eggs and the embryos were transferred into the pregnant rabbits. PCR, TA cloning and off-target analysis were performed on the F0 rabbits to identify whether the PCSK9 mutation was successful. Fifteen F0 rabbits were obtained. The sequencing results showed that one of them was PCSK9 point mutation homozygote and two of them were PCSK9 point mutation heterozygotes, and the mutation could be stably inherited. The rabbit model of PCSK9 point mutation was successfully constructed by CRISPR/Cas9 technique, which provides an animal model for exploring the molecular mechanism of impaired PCSK9 function and developing reliable and effective diagnosis and treatment measures.
Topics: Animals; CRISPR-Cas Systems; Humans; Point Mutation; Proprotein Convertase 9; Rabbits
PubMed: 34137224
DOI: 10.3724/zdxbyxb-2021-0133 -
ELife Dec 2022Together, copy-number and point mutations form the basis for most evolutionary novelty, through the process of gene duplication and divergence. While a plethora of...
Together, copy-number and point mutations form the basis for most evolutionary novelty, through the process of gene duplication and divergence. While a plethora of genomic data reveals the long-term fate of diverging coding sequences and their -regulatory elements, little is known about the early dynamics around the duplication event itself. In microorganisms, selection for increased gene expression often drives the expansion of gene copy-number mutations, which serves as a crude adaptation, prior to divergence through refining point mutations. Using a simple synthetic genetic reporter system that can distinguish between copy-number and point mutations, we study their early and transient adaptive dynamics in real time in . We find two qualitatively different routes of adaptation, depending on the level of functional improvement needed. In conditions of high gene expression demand, the two mutation types occur as a combination. However, under low gene expression demand, copy-number and point mutations are mutually exclusive; here, owing to their higher frequency, adaptation is dominated by copy-number mutations, in a process we term amplification hindrance. Ultimately, due to high reversal rates and pleiotropic cost, copy-number mutations may not only serve as a crude and transient adaptation, but also constrain sequence divergence over evolutionary time scales.
Topics: Point Mutation; Evolution, Molecular; Mutation; Adaptation, Physiological; Biological Evolution
PubMed: 36546673
DOI: 10.7554/eLife.82240 -
BMC Bioinformatics Oct 2022RNA deleterious point mutation prediction was previously addressed with programs such as RNAmute and MultiRNAmute. The purpose of these programs is to predict a global...
BACKGROUND
RNA deleterious point mutation prediction was previously addressed with programs such as RNAmute and MultiRNAmute. The purpose of these programs is to predict a global conformational rearrangement of the secondary structure of a functional RNA molecule, thereby disrupting its function. RNAmute was designed to deal with only single point mutations in a brute force manner, while in MultiRNAmute an efficient approach to deal with multiple point mutations was developed. The approach used in MultiRNAmute is based on the stabilization of the suboptimal RNA folding prediction solutions and/or destabilization of the optimal folding prediction solution of the wild type RNA molecule. The MultiRNAmute algorithm is significantly more efficient than the brute force approach in RNAmute, but in the case of long sequences and large m-point mutation sets the MultiRNAmute becomes exponential in examining all possible stabilizing and destabilizing mutations.
RESULTS
An inherent limitation in the RNAmute and MultiRNAmute programs is their ability to predict only substitution mutations, as these programs were not designed to work with deletion or insertion mutations. To address this limitation we herein develop a very fast algorithm, based on suboptimal folding solutions, to predict a predefined number of multiple point deleterious mutations as specified by the user. Depending on the user's choice, each such set of mutations may contain combinations of deletions, insertions and substitution mutations. Additionally, we prove the hardness of predicting the most deleterious set of point mutations in structural RNAs.
CONCLUSIONS
We developed a method that extends our previous MultiRNAmute method to predict insertion and deletion mutations in addition to substitutions. The additional advantage of the new method is its efficiency to find a predefined number of deleterious mutations. Our new method may be exploited by biologists and virologists prior to site-directed mutagenesis experiments, which involve indel mutations along with substitutions. For example, our method may help to investigate the change of function in an RNA virus via mutations that disrupt important motifs in its secondary structure.
Topics: INDEL Mutation; Mutation; Point Mutation; RNA; Sequence Analysis, RNA
PubMed: 36241988
DOI: 10.1186/s12859-022-04943-0 -
Stem Cell Research May 2021When studying patient specific induced pluripotent stem cells (iPS cells) as a disease model, the ideal control is an isogenic line that has corrected the point...
When studying patient specific induced pluripotent stem cells (iPS cells) as a disease model, the ideal control is an isogenic line that has corrected the point mutation, instead of iPS cells from siblings or other healthy subjects. However, repairing a point mutation in iPS cells even with the newly developed CRISPR-Cas9 technique remains difficult and time-consuming. Here we report a strategy that makes the Cas9 "knock-in" methodology both hassle-free and error-free. Instead of selecting a Cas9 recognition site close to the point mutation, we chose a site located in the nearest intron. We constructed a donor template with the fragment containing the corrected point mutation as one of the homologous recombination arms flanking a PGK-Puro cassette. After selection with puromycin, positive clones were identified and further transfected with a CRE vector to remove the PGK-Puro cassette. Using this methodology, we successfully repaired the point mutation G2019S of the LRRK2 gene in a Parkinson Disease (PD) patient iPS line and the point mutation R329H of the AARS1 gene in a Charcot-Marie-Tooth disease (CMT) patient iPS line. These isogenic iPS lines are ideal as a control in future studies.
Topics: CRISPR-Cas Systems; Clone Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Homologous Recombination; Humans; Induced Pluripotent Stem Cells; Mutation; Point Mutation
PubMed: 33857832
DOI: 10.1016/j.scr.2021.102332 -
Frontiers in Cellular and Infection... 2022Mutations may produce highly transmissible and damaging HIV variants, which increase the genetic diversity, and pose a challenge to develop vaccines. Therefore, it is of...
Mutations may produce highly transmissible and damaging HIV variants, which increase the genetic diversity, and pose a challenge to develop vaccines. Therefore, it is of great significance to understand how mutations drive the virulence of HIV. Based on the 11897 reliable genomes of HIV-1 retrieved from HIV sequence Database, we analyze the 12 types of point mutation (A>C, A>G, A>T, C>A, C>G, C>T, G>A, G>C, G>T, T>A, T>C, T>G) from multiple statistical perspectives for the first time. The global/geographical location/subtype/k-mer analysis results report that A>G, G>A, C>T and T>C account for nearly 64% among all SNPs, which suggest that APOBEC-editing and ADAR-editing may play an important role in HIV-1 infectivity. Time analysis shows that most genomes with abnormal mutation numbers comes from African countries. Finally, we use natural vector method to check the k-mer distribution changing patterns in the genome, and find that there is an important substitution pattern between nucleotides A and G, and 2-mer CG may have a significant impact on viral infectivity. This paper provides an insight into the single mutation of HIV-1 by using the latest data in the HIV sequence Database.
Topics: HIV-1; Point Mutation; Mutation; Mutation, Missense; Databases, Nucleic Acid
PubMed: 36457853
DOI: 10.3389/fcimb.2022.1033481 -
Cellular and Molecular Neurobiology Oct 2023The BAF (BRG1/BRM-associated factor) chromatin remodelling complex is essential for the regulation of DNA accessibility and gene expression during neuronal...
The BAF (BRG1/BRM-associated factor) chromatin remodelling complex is essential for the regulation of DNA accessibility and gene expression during neuronal differentiation. Mutations of its core subunit SMARCB1 result in a broad spectrum of pathologies, including aggressive rhabdoid tumours or neurodevelopmental disorders. Other mouse models have addressed the influence of a homo- or heterozygous loss of Smarcb1, yet the impact of specific non-truncating mutations remains poorly understood. Here, we have established a new mouse model for the carboxy-terminal Smarcb1 c.1148del point mutation, which leads to the synthesis of elongated SMARCB1 proteins. We have investigated its impact on brain development in mice using magnetic resonance imaging, histology, and single-cell RNA sequencing. During adolescence, Smarcb1 mice demonstrated rather slow weight gain and frequently developed hydrocephalus including enlarged lateral ventricles. In embryonic and neonatal stages, mutant brains did not differ anatomically and histologically from wild-type controls. Single-cell RNA sequencing of brains from newborn mutant mice revealed that a complete brain including all cell types of a physiologic mouse brain is formed despite the SMARCB1 mutation. However, neuronal signalling appeared disturbed in newborn mice, since genes of the AP-1 transcription factor family and neurite outgrowth-related transcripts were downregulated. These findings support the important role of SMARCB1 in neurodevelopment and extend the knowledge of different Smarcb1 mutations and their associated phenotypes.
Topics: Animals; Mice; Hydrocephalus; Mutation; Point Mutation; Signal Transduction; Transcription Factor AP-1
PubMed: 37219662
DOI: 10.1007/s10571-023-01361-5 -
Frontiers in Immunology 2022Major histocompatibility complex class II (MHC II) is an essential immune regulatory molecule that plays an important role in antigen presentation and T-cell...
Major histocompatibility complex class II (MHC II) is an essential immune regulatory molecule that plays an important role in antigen presentation and T-cell development. Abnormal MHC II expression can lead to immunodeficiency, clinically termed as type II bare lymphocyte syndrome (BLS), which usually results from mutations in the MHC II transactivator (CIITA) and other coactivators. Here, we present a new paradigm for MHC II deficiency in mice that involves a spontaneous point mutation on H2-Aa. A significantly reduced population of CD4 T cells was observed in mice obtained from the long-term homozygous breeding of (Map1, ) knockout mice; this phenotype was not attributed to the original knocked-out gene. MHC II expression was generally reduced, together with a marked deficiency of H2-Aa in the immune cells of these mice. Using cDNA and DNA sequencing, a spontaneous H2-Aa point mutation that led to false pre-mRNA splicing, deletion of eight bases in the mRNA, and protein frameshift was identified in these mice. These findings led to the discovery of a new type of spontaneous MHC II deficiency and provided a new paradigm to explain type II BLS in mice.
Topics: Animals; CD4-Positive T-Lymphocytes; Histocompatibility Antigens Class II; Mice; Mice, Knockout; Point Mutation; Severe Combined Immunodeficiency; T-Lymphocytes
PubMed: 35309308
DOI: 10.3389/fimmu.2022.810824