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 ecRESCUE: a novel ecDHFR-regulated RESCUE system with reduced RNA off-targeting activity

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Yihan Wang ◽  
Guo Li ◽  
Xiangyang Li ◽  
Yuzhe Wang ◽  
Xingxu Huang ◽  
...  
Keyword(s):  
Genetic Diseases ◽  
Transcriptional Level ◽  
In Vitro Experiments ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Considerable Potential ◽  
Base Editing ◽  
High Incidence ◽  
Rescue System

AbstractThe currently available RESCUE RNA base editing system demonstrates considerable potential for the treatment of genetic diseases at the transcriptional level. However, the relatively high incidence of off-target events hampers the precise RNA editing, thereby limiting its use in the clinical setting. This study describes a new RNA base editing method, named ecRESCUE, which utilizes inducible stabilization of the protein ecDHFR DD fused at the C-terminal of the original RESCUE system. In vitro experiments in 293T cells showed that the ecRESCUE editor markedly reduced the incidence of off-target single nucleotide polymorphisms without affecting the RNA A-to-I and C-to-U base editing efficiency. Altogether, these results demonstrate that the inducible ecRESCUE system represents an attractive approach to regulate and improve the outcome of the available RNA base editor with reduced off-targeting activity.

scholarly journals Base editing using CRISPR/Cas9 in Drosophila

2021 ◽  
Author(s):  
Elizabeth Marr ◽  
Christopher J. Potter
Keyword(s):  
Cytidine Deaminase ◽  
Point Mutations ◽  
Tissue Expression ◽  
Genomic Region ◽  
Target Sequence ◽  
Molecular Scaffolds ◽  
Base Editing ◽  
Guide Rna ◽  
Somatic Tissues ◽  
Transgenic Lines

AbstractCas9 and a guide RNA function to target specific genomic loci for generation of a double stranded break. Catalytic dead versions of Cas9 (dCas9) no longer cause double stranded breaks and instead can serve as molecular scaffolds to target additional enzymatic proteins to specific genomic loci. To generate mutations in selected genomic residues, dCas9 can be used for genomic base editing by fusing a cytidine deaminase to induce C>T (or G>A) mutations at targeted sites. Here, we test base editing in Drosophila by expressing a transgenic Drosophila base editor (DBE2, based on the mammalian BE2) which consists of a fusion protein of cytidine deaminase, dCas9, and uracil glycosylase inhibitor. We utilized transgenic lines expressing gRNAs along with pan-tissue expression of the Drosophila Base Editor (Actin5C-DBE2) and found high rates of base editing at multiple targeted loci in the 20 bp target sequence. Highest rates of conversion of C>T were found in positions 3-9 of the gRNA targeted site, with conversion reaching nearly 100% of targeted DNA is somatic tissues. The simultaneous use of two gRNA targeting a genomic region spaced ∼50 bps apart led to mutations between the two gRNA targets, implicating a method to broaden the available sites accessible to targeting. These results indicate base editing is efficient in Drosophila, and could be used to induce point mutations at select loci.

scholarly journals ecRESCUE: a novel ecDHFR-regulated RESCUE system with reduced RNA off-targeting activity

2021 ◽  
Author(s):  
Jianen Gao ◽  
Yihan Wang ◽  
Guo Li ◽  
Xiangyang Li ◽  
Yuzhe Wang ◽  
...  
Keyword(s):  
Genetic Diseases ◽  
Transcriptional Level ◽  
In Vitro Experiments ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Considerable Potential ◽  
Base Editing ◽  
High Incidence ◽  
Rescue System

Abstract The currently available RESCUE RNA base editing system demonstrates considerable potential for the treatment of genetic diseases at the transcriptional level. However, the relatively high incidence of off-target events hampers the precise RNA editing, thereby limiting its use in the clinical setting. This study describes a new RNA base editing method, named ecRESCUE, which utilizes inducible stabilization of the protein ecDHFR DD fused at the C-terminal of the original RESCUE system. In vitro experiments in 293T cells showed that the ecRESCUE editor markedly reduced the incidence of off-target single nucleotide polymorphisms without affecting the RNA A-to-I and C-to-U base editing efficiency. Altogether, these results demonstrate that the inducible ecRESCUE system represents an attractive approach to regulate and improve the outcome of the available RNA base editor with reduced off-targeting activity.

scholarly journals Efficient CRISPR-mediated base editing inAgrobacteriumspp.

2020 ◽  
Vol 118 (2) ◽  
pp. e2013338118
Author(s):  
Savio D. Rodrigues ◽  
Mansour Karimi ◽  
Lennert Impens ◽  
Els Van Lerberge ◽  
Griet Coussens ◽  
...  
Keyword(s):  
Hairy Root ◽  
Plant Pathogens ◽  
Cytidine Deaminase ◽  
Point Mutations ◽  
Loss Of Function ◽  
Base Editing ◽  
Causative Agents ◽  
Indispensable Tool ◽  
Mutation Strategies ◽  
Functional Gene Analysis

Agrobacteriumspp. are important plant pathogens that are the causative agents of crown gall or hairy root disease. Their unique infection strategy depends on the delivery of part of their DNA to plant cells. Thanks to this capacity, these phytopathogens became a powerful and indispensable tool for plant genetic engineering and agricultural biotechnology. AlthoughAgrobacteriumspp. are standard tools for plant molecular biologists, current laboratory strains have remained unchanged for decades and functional gene analysis ofAgrobacteriumhas been hampered by time-consuming mutation strategies. Here, we developed clustered regularly interspaced short palindromic repeats (CRISPR)-mediated base editing to enable the efficient introduction of targeted point mutations into the genomes of bothAgrobacterium tumefaciensandAgrobacterium rhizogenes. As an example, we generated EHA105 strains with loss-of-function mutations inrecA, which were fully functional for maize (Zea mays) transformation and confirmed the importance of RolB and RolC for hairy root development byA. rhizogenesK599. Our method is highly effective in 9 of 10 colonies after transformation, with edits in at least 80% of the cells. The genomes of EHA105 and K599 were resequenced, and genome-wide off-target analysis was applied to investigate the edited strains after curing of the base editor plasmid. The off-targets present were characteristic of Cas9-independent off-targeting and point to TC motifs as activity hotspots of the cytidine deaminase used. We anticipate that CRISPR-mediated base editing is the start of “engineering the engineer,” leading to improvedAgrobacteriumstrains for more efficient plant transformation and gene editing.

scholarly journals Next-generation cytosine base editors with minimized unguided DNA and RNA off-target events and high on-target activity

2020 ◽  
Author(s):  
Yi Yu ◽  
Thomas Leete ◽  
David A. Born ◽  
Lauren Young ◽  
Luis A. Barrera ◽  
...  
Keyword(s):  
Cytidine Deaminase ◽  
Point Mutations ◽  
Next Generation ◽  
Dna Double Strand Breaks ◽  
Cytosine Deamination ◽  
Base Editing ◽  
Dna And Rna ◽  
Fold Reduction ◽  
Target Dna ◽  
Target Activity

Abstract/introductory paragraphCytosine base editors (CBEs) are molecular machines which enable efficient and programmable reversion of T•A to C•G point mutations in the human genome without induction of DNA double strand breaks1, 2. Recently, the foundational cytosine base editor (CBE) ‘BE3’, containing rAPOBEC1, was reported to induce unguided, genomic DNA3, 4 and cellular RNA5 cytosine deamination when expressed in living cells. To mitigate spurious off-target events, we developed a sensitive, high-throughput cellular assay to select next-generation CBEs that display reduced spurious deamination profiles relative to rAPOBEC1-based CBEs, whilst maintaining equivalent or superior on-target editing frequencies. We screened 153 CBEs containing cytidine deaminase enzymes with diverse sequences and identified four novel CBEs with the most promising on/off target ratios. These spurious-deamination-minimized CBEs (BE4 with either RrA3F, AmAPOBEC1, SsAPOBEC3B, or PpAPOBEC1) were further optimized for superior on- and off-target DNA editing profiles through structure-guided mutagenesis of the deaminase domain. These next-generation CBEs display comparable overall DNA on-target editing frequencies, whilst eliciting a 10- to 49-fold reduction in C-to-U edits in the transcriptome of treated cells, and up to a 33-fold overall reduction in unguided off-target DNA deamination relative to BE4 containing rAPOBEC1. Taken together, these next-generation CBEs represent a new collection of base editing tools for applications in which minimization of spurious deamination is desirable and high on-target activity is required.

Recent Advances in CRISPR/Cas9 Directed Base Editing

2021 ◽  
Vol 21 ◽  
Author(s):  
Nan Liu ◽  
Lifang Zhou ◽  
Junyan , Qu ◽  
Shaohua Yao
Keyword(s):  
Recent Progress ◽  
High Efficiency ◽  
Genetic Diseases ◽  
Point Mutations ◽  
Clinical Applications ◽  
Great Promise ◽  
Base Editing ◽  
Recent Advances ◽  
Dna Base ◽  
Genomic Editing

: Recently, CRISPR based techniques had significantly improved our ability to make desired changes and regulations in various genomes. Among them, targeted base editing is one of the most powerful techniques in making precise genomic editing. Base editing enabled irreversible conversion of specific single DNA base, from C to T or and from A to G, in desired genomic loci. This technique has important implications in the study of human genetic diseases, considering that many of them resulted from point mutations. More importantly, high efficiency of those editing tools also provided great promise in clinical applications. In this review, we discuss recent progress and challenges of base editing tools.

scholarly journals Targeting specificity of APOBEC-based cytosine base editor in human iPSCs determined by whole genome sequencing

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Erica McGrath ◽  
Hyunsu Shin ◽  
Linyi Zhang ◽  
Je-Nie Phue ◽  
Wells W. Wu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Cytidine Deaminase ◽  
Point Mutations ◽  
Whole Genome ◽  
Human Stem Cells ◽  
Dna Double Strand Breaks ◽  
Base Editing ◽  
Human Ipscs

AbstractDNA base editors have enabled genome editing without generating DNA double strand breaks. The applications of this technology have been reported in a variety of animal and plant systems, however, their editing specificity in human stem cells has not been studied by unbiased genome-wide analysis. Here we investigate the fidelity of cytidine deaminase-mediated base editing in human induced pluripotent stem cells (iPSCs) by whole genome sequencing after sustained or transient base editor expression. While base-edited iPSC clones without significant off-target modifications are identified, this study also reveals the potential of APOBEC-based base editors in inducing unintended point mutations outside of likely in silico-predicted CRISPR-Cas9 off-targets. The majority of the off-target mutations are C:G->T:A transitions or C:G->G:C transversions enriched for the APOBEC mutagenesis signature. These results demonstrate that cytosine base editor-mediated editing may result in unintended genetic modifications with distinct patterns from that of the conventional CRISPR-Cas nucleases.

scholarly journals Precise and programmable C:G to G:C base editing in genomic DNA

2020 ◽  
Author(s):  
Liwei Chen ◽  
Jung Eun Park ◽  
Peter Paa ◽  
Priscilla D. Rajakumar ◽  
Yi Ting Chew ◽  
...  
Keyword(s):  
Therapeutic Potential ◽  
Excision Repair ◽  
Genetic Diseases ◽  
Cytosine Deaminase ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Base Editing ◽  
Guide Rnas ◽  
Base Excision ◽  
Nucleotide Resolution

AbstractMany genetic diseases are caused by single-nucleotide polymorphisms (SNPs). Base editors can correct SNPs at single-nucleotide resolution, but until recently, only allowed for C:G to T:A and A:T to G:C transition edits, addressing four out of twelve possible DNA base substitutions. Here we developed a novel class of C:G to G:C Base Editors (CGBEs) to create single-base genomic transversions in human cells. Our CGBEs consist of a nickase CRISPR-Cas9 (nCas9) fused to a cytosine deaminase and base excision repair (BER) proteins. Characterization of >30 CGBE candidates and 27 guide RNAs (gRNAs) revealed that CGBEs predominantly perform C:G to G:C editing (up to 90% purity), with rAPOBEC-nCas9-rXRCC1 being the most efficient (mean C:G to G:C edits at 15% and up to 37%). CGBEs target cytosine in WCW, ACC or GCT sequence contexts and within a precise two-nucleotide window of the target protospacer. We further targeted genes linked to dyslipidemia, hypertrophic cardiomyopathy, and deafness, showing the therapeutic potential of CGBE in interrogating and correcting human genetic diseases.

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 Programmable Base Editing in Mycobacterium tuberculosis Using an Engineered CRISPR RNA-Guided Cytidine Deaminase

2021 ◽  
Vol 3 ◽  
Author(s):  
Xin-Yuan Ding ◽  
Si-Shang Li ◽  
Yi-Man Geng ◽  
Mei-Yi Yan ◽  
Guo-Bao Li ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Mycobacterium Tuberculosis ◽  
Molecular Mechanisms ◽  
Cytidine Deaminase ◽  
Point Mutations ◽  
Treatment Strategies ◽  
Multidrug Resistant ◽  
Resistance Mechanisms ◽  
Bacterial Physiology ◽  
Base Editing

Multidrug-resistant Mycobacterium tuberculosis (Mtb) infection seriously endangers global human health, creating an urgent need for new treatment strategies. Efficient genome editing tools can facilitate identification of key genes and pathways involved in bacterial physiology, pathogenesis, and drug resistance mechanisms, and thus contribute to the development of novel treatments for drug-resistant tuberculosis. Here, we report a two-plasmid system, MtbCBE, used to inactivate genes and introduce point mutations in Mtb. In this system, the assistant plasmid pRecX-NucSE107A expresses RecX and NucSE107A to repress RecA-dependent and NucS-dependent DNA repair systems, and the base editor plasmid pCBE expresses a fusion protein combining cytidine deaminase APOBEC1, Cas9 nickase (nCas9), and uracil DNA glycosylase inhibitor (UGI). Together, the two plasmids enabled efficient G:C to A:T base pair conversion at desired sites in the Mtb genome. The successful development of a base editing system will facilitate elucidation of the molecular mechanisms underlying Mtb pathogenesis and drug resistance and provide critical inspiration for the development of base editing tools in other microbes.

Sign in / Sign up

Export Citation Format

Share Document