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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 -
International Journal of Molecular... Aug 2020Many genetic diseases and undesirable traits are due to base-pair alterations in genomic DNA. Base-editing, the newest evolution of clustered regularly interspaced short... (Review)
Review
Many genetic diseases and undesirable traits are due to base-pair alterations in genomic DNA. Base-editing, the newest evolution of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas-based technologies, can directly install point-mutations in cellular DNA without inducing a double-strand DNA break (DSB). Two classes of DNA base-editors have been described thus far, cytosine base-editors (CBEs) and adenine base-editors (ABEs). Recently, prime-editing (PE) has further expanded the CRISPR-base-edit toolkit to all twelve possible transition and transversion mutations, as well as small insertion or deletion mutations. Safe and efficient delivery of editing systems to target cells is one of the most paramount and challenging components for the therapeutic success of BEs. Due to its broad tropism, well-studied serotypes, and reduced immunogenicity, adeno-associated vector (AAV) has emerged as the leading platform for viral delivery of genome editing agents, including DNA-base-editors. In this review, we describe the development of various base-editors, assess their technical advantages and limitations, and discuss their therapeutic potential to treat debilitating human diseases.
Topics: CRISPR-Cas Systems; Dependovirus; Gene Editing; Genetic Predisposition to Disease; Humans; Point Mutation; RNA, Guide, CRISPR-Cas Systems; Tropism
PubMed: 32872311
DOI: 10.3390/ijms21176240 -
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 -
Cell Feb 2021Understanding the functional consequences of single-nucleotide variants is critical to uncovering the genetic underpinnings of diseases, but technologies to characterize...
Understanding the functional consequences of single-nucleotide variants is critical to uncovering the genetic underpinnings of diseases, but technologies to characterize variants are limiting. Here, we leverage CRISPR-Cas9 cytosine base editors in pooled screens to scalably assay variants at endogenous loci in mammalian cells. We benchmark the performance of base editors in positive and negative selection screens, identifying known loss-of-function mutations in BRCA1 and BRCA2 with high precision. To demonstrate the utility of base editor screens to probe small molecule-protein interactions, we screen against BH3 mimetics and PARP inhibitors, identifying point mutations that confer drug sensitivity or resistance. We also create a library of single guide RNAs (sgRNAs) predicted to generate 52,034 ClinVar variants in 3,584 genes and conduct screens in the presence of cellular stressors, identifying loss-of-function variants in numerous DNA damage repair genes. We anticipate that this screening approach will be broadly useful to readily and scalably functionalize genetic variants.
Topics: Alleles; BRCA1 Protein; BRCA2 Protein; Base Sequence; Catalytic Domain; Cell Line, Tumor; Gene Editing; Genetic Variation; High-Throughput Nucleotide Sequencing; Humans; Loss of Function Mutation; Mutagenesis; Myeloid Cell Leukemia Sequence 1 Protein; Point Mutation; Poly(ADP-ribose) Polymerases; Reproducibility of Results; Selection, Genetic; bcl-X Protein
PubMed: 33606977
DOI: 10.1016/j.cell.2021.01.012 -
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 -
Nature Communications Jul 2023Andes virus (ANDV) and Sin Nombre virus (SNV) are the etiologic agents of severe hantavirus cardiopulmonary syndrome (HCPS) in the Americas for which no FDA-approved...
Andes virus (ANDV) and Sin Nombre virus (SNV) are the etiologic agents of severe hantavirus cardiopulmonary syndrome (HCPS) in the Americas for which no FDA-approved countermeasures are available. Protocadherin-1 (PCDH1), a cadherin-superfamily protein recently identified as a critical host factor for ANDV and SNV, represents a new antiviral target; however, its precise role remains to be elucidated. Here, we use computational and experimental approaches to delineate the binding surface of the hantavirus glycoprotein complex on PCDH1's first extracellular cadherin repeat domain. Strikingly, a single amino acid residue in this PCDH1 surface influences the host species-specificity of SNV glycoprotein-PCDH1 interaction and cell entry. Mutation of this and a neighboring residue substantially protects Syrian hamsters from pulmonary disease and death caused by ANDV. We conclude that PCDH1 is a bona fide entry receptor for ANDV and SNV whose direct interaction with hantavirus glycoproteins could be targeted to develop new interventions against HCPS.
Topics: Animals; Cricetinae; Orthohantavirus; Point Mutation; Protocadherins; Communicable Diseases; Cadherins; Mesocricetus; RNA Viruses; Syndrome
PubMed: 37488123
DOI: 10.1038/s41467-023-40126-y -
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 -
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 -
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 -
MBio Jun 2023Meiosis is associated with genetic changes in the genome-via recombination, gene conversion, and mutations. The occurrence of gene conversion and mutations during...
Meiosis is associated with genetic changes in the genome-via recombination, gene conversion, and mutations. The occurrence of gene conversion and mutations during meiosis may further be influenced by the chromatin conformation, similar to the effect of the chromatin conformation on the mitotic mutation rate. To date, however, the exact distribution and type of meiosis-associated changes and the role of the chromatin conformation in this context are largely unexplored. Here, we determine recombination, gene conversion, and mutations using whole-genome sequencing of all meiotic products of 23 individual meioses in Zymoseptoria tritici, an important pathogen of wheat. We confirm a high genome-wide recombination rate of 65 centimorgan (cM)/Mb and see higher recombination rates on the accessory compared to core chromosomes. A substantial fraction of 0.16% of all polymorphic markers was affected by gene conversions, showing a weak GC-bias and occurring at higher frequency in regions of constitutive heterochromatin, indicated by the histone modification H3K9me3. The mutation rate associated with meiosis was approximately three orders of magnitude higher than the corresponding mitotic mutation rate. Importantly, repeat-induced point mutation (RIP), a fungal defense mechanism against duplicated sequences, is active in and responsible for the majority of these meiotic mutations. Our results indicate that the genetic changes associated with meiosis are a major source of variability in the genome of an important plant pathogen and shape its evolutionary trajectory. The impact of meiosis on the genome composition via gene conversion and mutations is mostly poorly understood, in particular, for non-model species. Here, we sequenced all four meiotic products for 23 individual meioses and determined the genetic changes caused by meiosis for the important fungal wheat pathogen Zymoseptoria tritici. We found a high rate of gene conversions and an effect of the chromatin conformation on gene conversion rates. Higher conversion rates were found in regions enriched with the H3K9me3-a mark for constitutive heterochromatin. Most importantly, meiosis was associated with a much higher frequency of mutations than mitosis; 78% of the meiotic mutations were caused by repeat-induced point mutations-a fungal defense mechanism against duplicated sequences. In conclusion, the genetic changes associated with meiosis are therefore a major factor shaping the genome of this fungal pathogen.
Topics: Gene Conversion; Point Mutation; Heterochromatin; Ascomycota; Mutation; Meiosis
PubMed: 37093087
DOI: 10.1128/mbio.03290-22