Abstract 55: Disruption of PCSK9 Using a Targeted Base Editing Strategy

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Alexandra C Chadwick ◽  
Kiran Musunuru
Keyword(s):  
Genome Editing ◽  
Rna Editing ◽  
Point Mutations ◽  
Hek293 Cells ◽  
Lower Efficiency ◽  
Nonsense Mutations ◽  
Base Editing ◽  
Stop Codons ◽  
Editing Domain

Proprotein convertase subtilisin/kexin type 9 (PCSK9) increases blood low-density lipoprotein (LDL) cholesterol by acting as an LDL receptor antagonist, thereby impairing LDL particle clearance. Since genetic disruption of PCSK9 is linked to reduced risk of coronary heart disease (CHD), our recent work has sought to permanently knock out the gene by using new genome editing technology. Recently reported “base editors” introduce point mutations at specific locations in the genome without the need for DNA double-strand breaks and, thus, with a lowered incidence of off-target effects. These base editors build on the CRISPR-Cas9 system by tethering an RNA-editing domain to a nickase version of Cas9, allowing for specific CT and/or GA base alterations. In this study, we used the “BE3” base editor (which uses the APOBEC-1 RNA-editing domain) to specifically target codons encoding tryptophans (TGG) or glutamines (CAG or CAA) to introduce nonsense mutations (producing stop codons TAG, TGA, or TAA) into human PCSK9 . The number of testable targets was increased by introducing specific point mutations into the BE3 construct (D1135V, R1335Q, T1337R in the Cas9 portion of BE3) to alter the protospacer adjacent motif (PAM) from NGG to NGA. Using HEK293 cells, we individually targeted a number of codons spanning the first seven exons in PCSK9 and identified several efficient targets. Most notably, the codons encoding glutamine 278 and glutamine 302 could be altered to stop codons in ~50% of alleles, as determined by the CEL-I nuclease mismatch assay and Sanger sequencing. We then targeted glutamine 278 in human induced pluripotent stem cells (iPSCs) and demonstrated base editing to introduce nonsense mutations into PCSK9 , albeit at a lower efficiency than in HEK293 cells. As a next step towards translation to human patients, we are targeting Pcsk9 in the mouse liver in vivo with BE3. Base editing may prove to be an efficient, safer strategy than standard CRISPR-Cas9 genome editing and holds promise as a strategy for the prevention of CHD.

scholarly journals Extending the Coding Potential of Viral Genomes with Overlapping Antisense ORFs: A Case for the De Novo Creation of the Gene Encoding the Antisense Protein ASP of HIV-1

Viruses ◽  
2022 ◽  
Vol 14 (1) ◽  
pp. 146
Author(s):  
Angelo Pavesi ◽  
Fabio Romerio
Keyword(s):  
De Novo ◽  
Point Mutations ◽  
Selective Advantage ◽  
Genomic Region ◽  
Fine Tuning ◽  
Sequence Evolution ◽  
Viral Genomes ◽  
Stop Codons ◽  
Hiv 1

Gene overprinting occurs when point mutations within a genomic region with an existing coding sequence create a new one in another reading frame. This process is quite frequent in viral genomes either to maximize the amount of information that they encode or in response to strong selective pressure. The most frequent scenario involves two different reading frames in the same DNA strand (sense overlap). Much less frequent are cases of overlapping genes that are encoded on opposite DNA strands (antisense overlap). One such example is the antisense ORF, asp in the minus strand of the HIV-1 genome overlapping the env gene. The asp gene is highly conserved in pandemic HIV-1 strains of group M, and it is absent in non-pandemic HIV-1 groups, HIV-2, and lentiviruses infecting non-human primates, suggesting that the ~190-amino acid protein that is expressed from this gene (ASP) may play a role in virus spread. While the function of ASP in the virus life cycle remains to be elucidated, mounting evidence from several research groups indicates that ASP is expressed in vivo. There are two alternative hypotheses that could be envisioned to explain the origin of the asp ORF. On one hand, asp may have originally been present in the ancestor of contemporary lentiviruses, and subsequently lost in all descendants except for most HIV-1 strains of group M due to selective advantage. Alternatively, the asp ORF may have originated very recently with the emergence of group M HIV-1 strains from SIVcpz. Here, we used a combination of computational and statistical approaches to study the genomic region of env in primate lentiviruses to shed light on the origin, structure, and sequence evolution of the asp ORF. The results emerging from our studies support the hypothesis of a recent de novo addition of the antisense ORF to the HIV-1 genome through a process that entailed progressive removal of existing internal stop codons from SIV strains to HIV-1 strains of group M, and fine tuning of the codon sequence in env that reduced the chances of new stop codons occurring in asp. Altogether, the study supports the notion that the HIV-1 asp gene encodes an accessory protein, providing a selective advantage to the virus.

scholarly journals ACBE, a new base editor for simultaneous C-to-T and A-to-G substitutions in mammalian systems

BMC Biology ◽  
2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Jingke Xie ◽  
Xingyun Huang ◽  
Xia Wang ◽  
Shixue Gou ◽  
Yanhui Liang ◽  
...  
Keyword(s):  
Cytidine Deaminase ◽  
Genetic Diseases ◽  
Point Mutations ◽  
Hek293 Cells ◽  
Multiple Point ◽  
Nucleotide Polymorphisms ◽  
Spacer Length ◽  
Endogenous Gene ◽  
Base Editing ◽  
Fetal Fibroblasts

Abstract Background Many favorable traits of crops and livestock and human genetic diseases arise from multiple single nucleotide polymorphisms or multiple point mutations with heterogeneous base substitutions at the same locus. Current cytosine or adenine base editors can only accomplish C-to-T (G-to-A) or A-to-G (T-to-C) substitutions in the windows of target genomic sites of organisms; therefore, there is a need to develop base editors that can simultaneously achieve C-to-T and A-to-G substitutions at the targeting site. Results In this study, a novel fusion adenine and cytosine base editor (ACBE) was generated by fusing a heterodimer of TadA (ecTadAWT/*) and an activation-induced cytidine deaminase (AID) to the N- and C-terminals of Cas9 nickase (nCas9), respectively. ACBE could simultaneously induce C-to-T and A-to-G base editing at the same target site, which were verified in HEK293-EGFP reporter cell line and 45 endogenous gene loci of HEK293 cells. Moreover, the ACBE could accomplish simultaneous point mutations of C-to-T and A-to-G in primary somatic cells (mouse embryonic fibroblasts and porcine fetal fibroblasts) in an applicable efficiency. Furthermore, the spacer length of sgRNA and the length of linker could influence the dual base editing activity, which provided a direction to optimize the ACBE system. Conclusion The newly developed ACBE would expand base editor toolkits and should promote the generation of animals and the gene therapy of genetic diseases with heterogeneous point mutations.

scholarly journals A qPCR method for genome editing efficiency determination and single-cell clone screening in human cells

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Bo Li ◽  
Naixia Ren ◽  
Lele Yang ◽  
Junhao Liu ◽  
Qilai Huang
Keyword(s):  
Single Cell ◽  
Genome Editing ◽  
Cell Clone ◽  
Editing Efficiency ◽  
Base Editing ◽  
Single Cell Clone ◽  
Cell Clones ◽  
Nucleotide Mismatch

AbstractCRISPR/Cas9 technology has been widely used for targeted genome modification both in vivo and in vitro. However, an effective method for evaluating genome editing efficiency and screening single-cell clones for desired modification is still lacking. Here, we developed this real time PCR method based on the sensitivity of Taq DNA polymerase to nucleotide mismatch at primer 3′ end during initiating DNA replication. Applications to CRISPR gRNAs targeting EMX1, DYRK1A and HOXB13 genes in Lenti-X 293 T cells exhibited comprehensive advantages. Just in one-round qPCR analysis using genomic DNA from cells underwent CRISPR/Cas9 or BE4 treatments, the genome editing efficiency could be determined accurately and quickly, for indel, HDR as well as base editing. When applied to single-cell clone screening, the genotype of each cell colony could also be determined accurately. This method defined a rigorous and practical way in quantify genome editing events.

scholarly journals Efficient and risk-reduced genome editing using double nicks enhanced by bacterial recombination factors in multiple species

2020 ◽  
Vol 48 (10) ◽  
pp. e57-e57
Author(s):  
Xiaozhen He ◽  
Wenfeng Chen ◽  
Zhen Liu ◽  
Guirong Yu ◽  
Youbang Chen ◽  
...  
Keyword(s):  
Genome Editing ◽  
Human Peripheral Blood ◽  
Point Mutations ◽  
Peripheral Blood Lymphocytes ◽  
Dna Double Strand Breaks ◽  
Site Specific ◽  
Low Efficiency ◽  
Multiple Species ◽  
Balancing Efficiency

Abstract Site-specific DNA double-strand breaks have been used to generate knock-in through the homology-dependent or -independent pathway. However, low efficiency and accompanying negative impacts such as undesirable indels or tumorigenic potential remain problematic. In this study, we present an enhanced reduced-risk genome editing strategy we named as NEO, which used either site-specific trans or cis double-nicking facilitated by four bacterial recombination factors (RecOFAR). In comparison to currently available approaches, NEO achieved higher knock-in (KI) germline transmission frequency (improving from zero to up to 10% efficiency with an average of 5-fold improvement for 8 loci) and ‘cleaner’ knock-in of long DNA fragments (up to 5.5 kb) into a variety of genome regions in zebrafish, mice and rats. Furthermore, NEO yielded up to 50% knock-in in monkey embryos and 20% relative integration efficiency in non-dividing primary human peripheral blood lymphocytes (hPBLCs). Remarkably, both on-target and off-target indels were effectively suppressed by NEO. NEO may also be used to introduce low-risk unrestricted point mutations effectively and precisely. Therefore, by balancing efficiency with safety and quality, the NEO method reported here shows substantial potential and improves the in vivo gene-editing strategies that have recently been developed.

Recent advances in DNA-free editing and precise base editing in plants

2017 ◽  
Vol 1 (2) ◽  
pp. 161-168 ◽  
Author(s):  
Yi Zhang ◽  
Caixia Gao
Keyword(s):  
Genome Editing ◽  
Genome Engineering ◽  
Point Mutations ◽  
Plant Genome ◽  
The Novel ◽  
Future Perspectives ◽  
Delivery Methods ◽  
Base Editing ◽  
Short Palindromic Repeat ◽  
Target Effects

Genome-editing technologies based on the CRISPR (clustered regularly interspaced short palindromic repeat) system have been widely used in plants to investigate gene function and improve crop traits. The recently developed DNA-free delivery methods and precise base-editing systems provide new opportunities for plant genome engineering. In this review, we describe the novel DNA-free genome-editing methods in plants. These methods reduce off-target effects and may alleviate regulatory concern about genetically modified plants. We also review applications of base-editing systems, which are highly effective in generating point mutations and are of great value for introducing agronomically valuable traits. Future perspectives for DNA-free editing and base editing are also discussed.

scholarly journals IDH1 Splicing Alterations in Patients with AML and Their Relationship to Blood 2HG Levels

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1060-1060
Author(s):  
Sophia Adamia ◽  
Patricia Toniolo ◽  
Amir T. Fathi ◽  
Ilene Galinsky ◽  
Suiyang Liu ◽  
...  
Keyword(s):  
Splice Variant ◽  
Splice Variants ◽  
Point Mutations ◽  
Advisory Board ◽  
Blood Levels ◽  
Sequencing Analysis ◽  
Isocitrate Dehydrogenase 1 ◽  
Nested Polymerase Chain Reaction ◽  
Stop Codons

Abstract Introduction: Point mutations in the human cytosolic isocitrate dehydrogenase 1 (IDH1) gene, or its mitochondrial homolog IDH2 gene, are frequent in patients with AML, particularly at residues R132 in IDH1 and, R140 or R172 in IDH2. These mutations cause loss of the enzymeÕs ability to catalyze the conversion of isocitrate to α-ketoglutarate and result in neomorphic production of R(-)-2-hydroxyglutarate (2HG). It has been reported that 2HG functions as an ÒoncometaboliteÓ. Interestingly, serum levels of 2HG are highly variable among patients with the same mutations, and occasionally are elevated even in patients without detectable mutations. Previously Chaturvedi et al (ASH 2011, Abstract) showed that an IDH1 splice variant promotes leukemogenesis in vivo in the absence of IDH1/IDH2 mutations. In this study, we examined the frequency of this splice variant and two other IDH1 novel splice variants in patients with AML, and looked for any relationships between splice variants, mutations, and 2HG levels. Methods: Cloning and sequencing analysis were used to identify IDH1 and IDH2 splice variants. DNA fragment analysis and capillary electrophoresis was used to evaluate expression of splice variants in 242 patients, of which 166 patients had 2HG blood levels measured. As controls we used two CD34+ bone marrow (BM) cells, three un-fractionated BM cells, and purified neutrophil and monocytes from three peripheral blood samples (PB) of normal donors. To detect point mutations, DNA fragments spanning exon 4 of IDH1 and IDH2 were amplified by nested polymerase chain reaction, and then PCR products were directly sequenced by Sanger sequencing. Results: We studied three splice variants of IDH1: IDH1Va (reported previously), and two variants discovered in the course of this study, IDH1Vb and IDH1Vc. Alignment analyses of these splice variants indicated that the IDH1Vb variant transcripts do not cause a frame shift, and retain native start and stop codons, while IDH1Va and IDH1Vc transcripts have frame shifts that create premature stop codons in the last exon. No splice variants of IDH2 were identified in these patient samples. Of the 242 AML samples (PB-166, BM-76) 51% expressed IDH1Va, 49% expressed IDH1Vb and 23% expressed IDHVc, almost always in combination with full length of IDH1 transcripts (IDH1FL). These IDH1 splice variants were not detected in the normal cells analyzed. We compared expression of IDH1 splice variants, presence of common point mutations, and levels of 2-HG in the 166 patients (Table 1). As expected, patients with point mutations in IDH1 or IDH2 generally had high levels of 2HG in their blood (> average 6104 ng/ml). Seven patients had detectable IDH1/IDH2 point mutations but low 2-HG (< average 222 ng/ml) blood levels. In these patients IDH1Va and IDH1Vb splice variants were detected frequently; Five of 7 patients expressed these variants together and/or in combination with the IDH1-FL mRNA. In contrast, 14 patients had no detectable IDH1/IDH2 mutations but elevated (> average 4430 ng/ml) 2-HG blood levels, twelve of these overexpressed the IDH1Vb splice variant transcripts (Table 1). Six of these 12 patients expressed only IDH1Vb transcripts together with IDH1FL; the average 2HG blood levels in these patients was 2875ng/ml. Conclusion: Though high 2-HG blood levels are correlated with the presence of IDH1/IDH2 mutations, the degree of 2-HG elevation may be altered by other events such as alternative splicing. The expression of IDH1 splice variants was commonly detected in AML samples. Studies to examine the effects of splice variants of IDH1 on 2HG production and transformation are underway. Figure 1 Figure 1. Disclosures Fathi: Millennium: Research Funding; Seattle Genetics: Advisory Board, Advisory Board Other; Agios: Advisory Board, Advisory Board Other. Yen:Agios: Employment. Kim:Agios: Employment. Zhu:Agios Pharmaceuticals: Employment, Stockholder Other. Kim:Yes, same as for being Author of Abstract #70656 Hide Full Conflict-of-Interest Disclosure I have relevant financial relationship(s) to disclose. Yes Name of Organization Type of relationship Agios Pharmaceuticals: Employment. Steensma:Novartis: Consultancy; Celgene: Consultancy; Ariad: Equity Ownership; Amgen: Consultancy. Stone:Agios: Consultancy.

scholarly journals Efficient precise in vivo base editing in adult dystrophic mice

2020 ◽  
Author(s):  
Li Xu ◽  
Chen Zhang ◽  
Haiwen Li ◽  
Peipei Wang ◽  
Yandi Gao ◽  
...  
Keyword(s):  
Rna Editing ◽  
Deep Sequencing ◽  
High Efficiency ◽  
Contractile Function ◽  
Functional Improvement ◽  
Base Editing ◽  
Monogenic Diseases ◽  
Editing Activity ◽  
Target Rna

ABSTRACTBackgroundRecent advances in the base editing technology have created an exciting opportunity to precisely correct disease-causing mutations. However, the large size of base editors and their inherited off-target activities pose challenges for in vivo base editing. Moreover, the requirement of a protospacer adjacent motif (PAM) sequence within a suitable window near the mutation site further limits the targeting feasibility. In this work, we rationally improved the adenine base editor (ABE) to overcome these challenges and demonstrated the exceptionally high efficiency to precisely edit the Duchenne muscular dystrophy (DMD) mutation in adult mice.MethodsWe employed a fluorescence reporter assay to assess the feasibility of ABE to correct the dystrophin mutation in mdx4cv mice. The intein protein trans-splicing (PTS) was used to split the oversized ABE into two halves for efficient packaging into adeno-associated virus 9 (AAV9). The ABE with broadened PAM recognition (ABE-NG) was rationally re-designed for improved off-target RNA editing activity and on-target DNA editing efficiency. The mdx4cv mice at the 5 weeks of age receiving intramuscular or intravenous injections of AAV9 carrying the improved ABE-NG were analyzed at 10 weeks or 10 months of age. The editing outcomes were analyzed by Sanger and deep sequencing of the amplicons, immunofluorescence staining, Western blot and contractile function measurements. The off-target activities, host immune response and long-term toxicity were analyzed by deep sequencing, ELISA and serological assays, respectively.ResultsWe showed efficient in vitro base correction of the dystrophin mutation carried in mdx4cv mice using ABE-NG. The super-fast intein-splits of ABE-NG enabled the expression of full-length ABE-NG and efficient AAV9 packaging. We rationally improved ABE-NG with eliminated off-target RNA editing activity and minimal PAM requirement, and packaged into AAV9 (AAV9-iNG). Intramuscular and intravenous administration of AAV9-iNG resulted in dystrophin restoration and functional improvement. At 10 months after AAV9-iNG treatment, a near complete rescue of dystrophin was measured in mdx4cv mouse hearts. The off-target activities remained low and no obvious toxicity was detected.ConclusionsThis study highlights the promise of permanent base editing using iABE-NG for the treatment of monogenic diseases, in particular, the genetic cardiomyopathies.

scholarly journals Prediction of synonymous corrections by the BE-FF computational tool expands the targeting scope of base editing

2020 ◽  
Author(s):  
Rabinowitz Roy ◽  
Abadi Shiran ◽  
Almog Shiri ◽  
Offen Daniel
Keyword(s):  
Genome Editing ◽  
Point Mutations ◽  
Nucleotide Variation ◽  
Single Nucleotide Variation ◽  
Computational Tool ◽  
Single Nucleotide ◽  
Base Editing ◽  
A Genome ◽  
Wide Range ◽  
Reference Protein

ABSTRACTBase editing is a genome-editing approach that employs the CRISPR/Cas system to precisely install point mutations within the genome. A cytidine or adenosine deaminase enzyme is fused to a deactivated Cas and converts C to T or A to G, respectively. The diversified repertoire of base editors, varied in their Cas and deaminase proteins, provides a wide range of functionality. However, existing base-editors can only induce transition substitutions in a specified region determined by the base editor, thus, they are incompatible for many point mutations. Here, we present BE-FF (Base Editors Functional Finder), a novel computational tool that identifies suitable base editors to correct the translated sequence erred by a given single nucleotide variation. Even if a perfect correction of the single nucleotide variation is not possible, BE-FF detects synonymous corrections to produce the reference protein. To assess the potential of BE-FF, we analysed a database of human pathogenic point mutations and found suitable base editors for 60.9% of the transition mutations. Importantly, 19.4% of them were made possible only by synonymous corrections. Moreover, we detected 298 cases in which pathogenic mutations caused by transversions were potentially repairable by base editing via synonymous corrections, although it had been thought impractical. The BE-FF tool and the database are available at https://www.danioffenlab.com/be-ff.GRAPHICAL ABSTRACT

scholarly journals Precise base editing for the in vivo study of developmental signaling and human pathologies in zebrafish

2020 ◽  
Author(s):  
Marion Rosello ◽  
Juliette Vougny ◽  
François Czarny ◽  
Marina Mione ◽  
Jean-Paul Concordet ◽  
...  
Keyword(s):  
High Efficiency ◽  
Human Gene ◽  
Point Mutations ◽  
Base Change ◽  
Model System ◽  
Human Genetic Disease ◽  
Base Editing ◽  
Oncogenic Mutations ◽  
Cell Signaling Pathways

While zebrafish is emerging as a new model system to study human diseases, an efficient methodology to generate precise point mutations at high efficiency is still lacking. Here we show that base editors can generate C-to-T point mutations with high efficiencies without other unwanted on-target mutations. In addition, we established a new editor variant recognizing an NAA PAM, expanding the base editing possibilities in zebrafish. Using these approaches, we first generated a base change in the ctnnb1 gene, mimicking oncogenic mutations of the human gene known to result in constitutive activation of endogenous Wnt signaling. Additionally, we precisely targeted several cancer-associated genes among which cbl. With this last target we created a new zebrafish dwarfism model. Together our findings expand the potential of zebrafish as a model system allowing new approaches for the endogenous modulation of cell signaling pathways and the generation of precise models of human genetic disease associated-mutations.

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