-
Methods in Molecular Biology (Clifton,... 2017Inverse PCR is a powerful tool for the rapid introduction of desired mutations at desired positions in a circular double-stranded DNA sequence. Here, custom-designed...
Inverse PCR is a powerful tool for the rapid introduction of desired mutations at desired positions in a circular double-stranded DNA sequence. Here, custom-designed mutant primers oriented in the inverse direction are used to amplify the entire circular template with incorporation of the required mutation(s). By careful primer design it can be used to perform such diverse modifications as the introduction of point mutations and multiple mutations, the insertion of new sequences, and even sequence deletions. Three primer formats are commonly used; nonoverlapping, partially overlapping and fully overlapping primers, and here we describe the use of nonoverlapping primers for introduction of a point mutation. Use of such a primer setup in the PCR reaction, with one of the primers containing the desired mismatch mutation, results in the amplification of a linear, double-stranded, mutated product. Methylated template DNA is removed from the nonmethylated PCR product by DpnI digestion and the PCR product is then phosphorylated by polynucleotide kinase treatment before being recircularized by ligation, and transformed to E. coli. This relatively simple site-directed mutagenesis procedure is of major importance in biology and biotechnology today where it is commonly employed for the study and engineering of DNA, RNA, and proteins.
Topics: DNA; DNA Primers; Mutagenesis, Site-Directed; Point Mutation; Polymerase Chain Reaction
PubMed: 28540701
DOI: 10.1007/978-1-4939-7060-5_5 -
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 -
Methods in Molecular Biology (Clifton,... 2019Current genome editing tools enable targeted mutagenesis of selected DNA sequences in many species. However, the efficiency and the type of introduced mutations by the...
Current genome editing tools enable targeted mutagenesis of selected DNA sequences in many species. However, the efficiency and the type of introduced mutations by the genome editing method are largely dependent on the target site. As a consequence, the outcome of the editing operation is difficult to predict. Therefore, a quick assay to quantify the frequency of mutations is vital for a proper assessment of genome editing actions. We developed two methods that are rapid, cost-effective, and readily applicable: (1) TIDE, which can accurately identify and quantify insertions and deletions (indels) that arise after introduction of double strand breaks (DSBs); (2) TIDER, which is suited for template-mediated editing events including point mutations. Both methods only require a set of PCR reactions and standard Sanger sequencing runs. The sequence traces are analyzed by the TIDE or TIDER algorithm (available at https://tide.nki.nl or https://deskgen.com ). The routine is easy, fast, and provides much more detailed information than current enzyme-based assays. TIDE and TIDER accelerate testing and designing of DSB-based genome editing strategies.
Topics: Clustered Regularly Interspaced Short Palindromic Repeats; DNA Breaks, Double-Stranded; Gene Editing; INDEL Mutation; Mutagenesis; Mutation; Point Mutation
PubMed: 30912038
DOI: 10.1007/978-1-4939-9170-9_3 -
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 -
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 -
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 -
Advances in Genetics 2020
Topics: Animals; Biological Evolution; Earth, Planet; Humans; Origin of Life; Point Mutation
PubMed: 33081929
DOI: 10.1016/S0065-2660(20)30036-5 -
International Journal of Molecular... Dec 2022Genetic aberrations, including chromosomal rearrangements, loss or amplification of DNA, and point mutations, are major elements of cancer development [...].
Genetic aberrations, including chromosomal rearrangements, loss or amplification of DNA, and point mutations, are major elements of cancer development [...].
Topics: Humans; Epigenesis, Genetic; Chromosome Aberrations; Neoplasms; Point Mutation; DNA
PubMed: 36555088
DOI: 10.3390/ijms232415446 -
World Journal of Microbiology &... Jul 2020The fermentation of industrial bacteria encounters a serious problem in continuous culture, i.e. the production traits lose. However, current research on the mechanism... (Review)
Review
The fermentation of industrial bacteria encounters a serious problem in continuous culture, i.e. the production traits lose. However, current research on the mechanism of strain degeneration is not clear enough, and there are few methods to effectively control the degeneration. Under growth restriction, the mutation rate of fermentation strains increases. Many cellular processes and poor fermentation conditions can trigger the transposition of transposable elements, SOS response, and RpoS-controlled adaptive mutations, causing genetic instability. Genetic instability which resulted from point mutations and genomic rearrangements can be responsible for strain degeneration. This mini-review summarizes the degeneration phenomena and mechanisms in common industrial bacteria and highlights three mechanisms of strain degeneration, including the transposition of transposable elements, SOS response, and adaptive mutations. According to different mutation mechanisms, many promising strategies have been proposed to increase the stability and the yield of industrial strains, for example, developing platform strains free of insertion sequence to enhance the stability of recombinant plasmid, using SOS inhibitors to block the SOS response, and improving environmental tolerance capacity and fermentation conditions to reduce adaptive mutations.
Topics: DNA Transposable Elements; Fermentation; Genomic Instability; Industrial Microbiology; Mutation; Phenotype; Point Mutation; Recombination, Genetic
PubMed: 32681370
DOI: 10.1007/s11274-020-02901-7 -
Microbiology Spectrum Jul 2017Transposable elements have colonized the genomes of nearly all organisms, including fungi. Although transposable elements may sometimes provide beneficial functions to... (Review)
Review
Transposable elements have colonized the genomes of nearly all organisms, including fungi. Although transposable elements may sometimes provide beneficial functions to their hosts their overall impact is considered deleterious. As a result, the activity of transposable elements needs to be counterbalanced by the host genome defenses. In fungi, the primary genome defense mechanisms include repeat-induced point mutation (RIP) and methylation induced premeiotically, meiotic silencing by unpaired DNA, sex-induced silencing, cosuppression (also known as somatic quelling), and cotranscriptional RNA surveillance. Recent studies of the filamentous fungus have shown that the process of repeat recognition for RIP apparently involves interactions between coaligned double-stranded segments of chromosomal DNA. These studies have also shown that RIP can be mediated by the conserved pathway that establishes transcriptional (heterochromatic) silencing of repetitive DNA. In light of these new findings, RIP emerges as a specialized case of the general phenomenon of heterochromatic silencing of repetitive DNA.
Topics: DNA Transposable Elements; DNA, Fungal; Fungi; Genome, Fungal; Meiosis; Point Mutation; Repetitive Sequences, Nucleic Acid
PubMed: 28721856
DOI: 10.1128/microbiolspec.FUNK-0042-2017