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Nature Dec 2019Most genetic variants that contribute to disease are challenging to correct efficiently and without excess byproducts. Here we describe prime editing, a versatile and...
Most genetic variants that contribute to disease are challenging to correct efficiently and without excess byproducts. Here we describe prime editing, a versatile and precise genome editing method that directly writes new genetic information into a specified DNA site using a catalytically impaired Cas9 endonuclease fused to an engineered reverse transcriptase, programmed with a prime editing guide RNA (pegRNA) that both specifies the target site and encodes the desired edit. We performed more than 175 edits in human cells, including targeted insertions, deletions, and all 12 types of point mutation, without requiring double-strand breaks or donor DNA templates. We used prime editing in human cells to correct, efficiently and with few byproducts, the primary genetic causes of sickle cell disease (requiring a transversion in HBB) and Tay-Sachs disease (requiring a deletion in HEXA); to install a protective transversion in PRNP; and to insert various tags and epitopes precisely into target loci. Four human cell lines and primary post-mitotic mouse cortical neurons support prime editing with varying efficiencies. Prime editing shows higher or similar efficiency and fewer byproducts than homology-directed repair, has complementary strengths and weaknesses compared to base editing, and induces much lower off-target editing than Cas9 nuclease at known Cas9 off-target sites. Prime editing substantially expands the scope and capabilities of genome editing, and in principle could correct up to 89% of known genetic variants associated with human diseases.
Topics: Cell Line; DNA; DNA Breaks, Double-Stranded; Gene Editing; Genome; Humans; Point Mutation; Saccharomyces cerevisiae
PubMed: 31634902
DOI: 10.1038/s41586-019-1711-4 -
Cell Nov 2017Cancer develops as a result of somatic mutation and clonal selection, but quantitative measures of selection in cancer evolution are lacking. We adapted methods from...
Cancer develops as a result of somatic mutation and clonal selection, but quantitative measures of selection in cancer evolution are lacking. We adapted methods from molecular evolution and applied them to 7,664 tumors across 29 cancer types. Unlike species evolution, positive selection outweighs negative selection during cancer development. On average, <1 coding base substitution/tumor is lost through negative selection, with purifying selection almost absent outside homozygous loss of essential genes. This allows exome-wide enumeration of all driver coding mutations, including outside known cancer genes. On average, tumors carry ∼4 coding substitutions under positive selection, ranging from <1/tumor in thyroid and testicular cancers to >10/tumor in endometrial and colorectal cancers. Half of driver substitutions occur in yet-to-be-discovered cancer genes. With increasing mutation burden, numbers of driver mutations increase, but not linearly. We systematically catalog cancer genes and show that genes vary extensively in what proportion of mutations are drivers versus passengers.
Topics: Humans; INDEL Mutation; Microsatellite Instability; Models, Genetic; Mutation Rate; Neoplasms; Point Mutation; Polymorphism, Single Nucleotide; Selection, Genetic
PubMed: 29056346
DOI: 10.1016/j.cell.2017.09.042 -
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 -
Biomolecules Feb 2023Sickle cell disease is the consequence of a single point mutation on the surface of the β chains of the hemoglobin molecule leading to the formation of rigid polymers... (Review)
Review
Sickle cell disease is the consequence of a single point mutation on the surface of the β chains of the hemoglobin molecule leading to the formation of rigid polymers that disrupt circulation. It has long been established that the polymers are comprised of seven pairs of double strands that are twisted replicas of the double strands found in crystals. Here, we review several newer developments that elaborate on that simple model and provide deeper insights into the process.
Topics: Humans; Hemoglobin, Sickle; Anemia, Sickle Cell; Hemoglobins; Polymers; Point Mutation
PubMed: 36979347
DOI: 10.3390/biom13030413 -
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 -
Microbiology Spectrum Apr 2022Mutant spectra of RNA viruses are important to understand viral pathogenesis and response to selective pressures. There is a need to characterize the complexity of...
Mutant spectra of RNA viruses are important to understand viral pathogenesis and response to selective pressures. There is a need to characterize the complexity of mutant spectra in coronaviruses sampled from infected patients. In particular, the possible relationship between SARS-CoV-2 mutant spectrum complexity and disease associations has not been established. In the present study, we report an ultradeep sequencing (UDS) analysis of the mutant spectrum of amplicons from the nsp12 (polymerase)- and spike (S)-coding regions of 30 nasopharyngeal isolates (diagnostic samples) of SARS-CoV-2 of the first COVID-19 pandemic wave (Madrid, Spain, April 2020) classified according to the severity of ensuing COVID-19. Low-frequency mutations and deletions, counted relative to the consensus sequence of the corresponding isolate, were overwhelmingly abundant. We show that the average number of different point mutations, mutations per haplotype, and several diversity indices was significantly higher in SARS-CoV-2 isolated from patients who developed mild disease than in those associated with moderate or severe disease (exitus). No such bias was observed with RNA deletions. Location of amino acid substitutions in the three-dimensional structures of nsp12 (polymerase) and S suggest significant structural or functional effects. Thus, patients who develop mild symptoms may be a richer source of genetic variants of SARS-CoV-2 than patients with moderate or severe COVID-19. The study shows that mutant spectra of SARS-CoV-2 from diagnostic samples differ in point mutation abundance and complexity and that significantly larger values were observed in virus from patients who developed mild COVID-19 symptoms. Mutant spectrum complexity is not a uniform trait among isolates. The nature and location of low-frequency amino acid substitutions present in mutant spectra anticipate great potential for phenotypic diversification of SARS-CoV-2.
Topics: COVID-19; Humans; Mutation; Nasopharynx; Pandemics; Point Mutation; SARS-CoV-2
PubMed: 35348367
DOI: 10.1128/spectrum.00221-22 -
Current Opinion in Pharmacology Oct 2016Improvements in databases have already impacted GPCR research. The purpose of the review is to give a snapshot of the GPCR data available and provide utility examples.... (Review)
Review
Improvements in databases have already impacted GPCR research. The purpose of the review is to give a snapshot of the GPCR data available and provide utility examples. Consequently, this review covers a small set of major databases, including UniProt for proteins, Ensembl for genes, ChEMBL for bioactive chemistry and SureChEMBL for patents. In addition, two portals are outlined, GPCRdb and the IUPHAR/BPS Guide to PHARMACOLOGY (GtoPdb) that are based on expert annotation. The former has an emphasis on structures, sequences, point mutations, analysis tools and visualisation. The latter focuses on endogenous GPCR ligands, pharmacological modulation, approved drugs, clinical candidates and tool compounds. Since data growth is accelerating, those embarking on GPCR projects should not only check databases but also recent journal and patent publications.
Topics: Databases, Factual; Drug Discovery; Humans; Ligands; Pharmaceutical Preparations; Point Mutation; Receptors, G-Protein-Coupled
PubMed: 27472010
DOI: 10.1016/j.coph.2016.07.002 -
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 -
Science Bulletin Dec 2023Gene editing ushers in a new era of disease treatment since many genetic diseases are caused by base-pair mutations in genomic DNA. With the rapid development of genome... (Review)
Review
Gene editing ushers in a new era of disease treatment since many genetic diseases are caused by base-pair mutations in genomic DNA. With the rapid development of genome editing technology, novel editing tools such as base editing and prime editing (PE) have attracted public attention, heralding a great leap forward in this field. PE, in particular, is characterized by no need for double-strand breaks (DSBs) or homology sequence templates with variable application scenarios, including point mutations as well as insertions or deletions. With higher editing efficiency and fewer byproducts than traditional editing tools, PE holds great promise as a therapeutic strategy for human diseases. Subsequently, a growing demand for the standard construction of PE system has spawned numerous easy-to-access internet resources and tools for personalized prime editing guide RNA (pegRNA) design and off-target site prediction. In this review, we mainly introduce the innovation and evolutionary strategy of PE systems and the auxiliary tools for PE design and analysis. Additionally, its application and future potential in the clinical field have been summarized and envisaged.
Topics: Humans; CRISPR-Cas Systems; RNA, Guide, CRISPR-Cas Systems; Gene Editing; Mutation; Point Mutation
PubMed: 37973465
DOI: 10.1016/j.scib.2023.11.015 -
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