scholarly journals A single CRISPR base editor to induce simultaneous C-to-T and A-to-G mutations

2019 ◽  
Author(s):  
Rina C. Sakata ◽  
Soh Ishiguro ◽  
Hideto Mori ◽  
Mamoru Tanaka ◽  
Motoaki Seki ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Cytidine Deaminase ◽  
Sequencing Data ◽  
Base Editing ◽  
Dna Molecule ◽  
Deep Sequencing Data ◽  
Target Dna ◽  
Activation Induced Cytidine Deaminase ◽  
Target Sites ◽  
Sequence Window

While several Cas9-derived base editors have been developed to induce either C-to-T or A-to-G mutation at target genomic sites, the possible genome editing space when using the current base editors remains limited. Here, we present a novel base editor, Target-ACE, which integrates the abilities of both of the previously developed C-to-T and A-to-G base editors by fusing an activation-induced cytidine deaminase (AID) and an engineered tRNA adenosine deaminase (TadA) to a catalytically impaired Streptococcus pyogenes Cas9. In mammalian cells, Target-ACE enabled heterologous editing of multiple bases in a small sequence window of target sites with increased efficiency compared with a mixture of two relevant base editor enzymes, each of which may block the same target DNA molecule from the other. Furthermore, by modeling editing patterns using deep sequencing data, the editing spectra of Target-ACE and other base editors were simulated across the human genome, demonstrating the highest potency of Target-ACE to edit amino acid coding patterns. Taking these findings together, Target-ACE is a new tool that broadens the capabilities for base editing for various applications.

scholarly journals Engineered CRISPR-Cas9 nuclease with expanded targeting space

Science ◽  
2018 ◽  
Vol 361 (6408) ◽  
pp. 1259-1262 ◽  
Author(s):  
Hiroshi Nishimasu ◽  
Xi Shi ◽  
Soh Ishiguro ◽  
Linyi Gao ◽  
Seiichi Hirano ◽  
...  
Keyword(s):  
Cytidine Deaminase ◽  
Specific Interaction ◽  
Human Cells ◽  
The Third ◽  
Cas9 Nuclease ◽  
Protospacer Adjacent Motif ◽  
Target Dna ◽  
Activation Induced Cytidine Deaminase ◽  
Target Sites ◽  
Endogenous Target

The RNA-guided endonuclease Cas9 cleaves its target DNA and is a powerful genome-editing tool. However, the widely used Streptococcus pyogenes Cas9 enzyme (SpCas9) requires an NGG protospacer adjacent motif (PAM) for target recognition, thereby restricting the targetable genomic loci. Here, we report a rationally engineered SpCas9 variant (SpCas9-NG) that can recognize relaxed NG PAMs. The crystal structure revealed that the loss of the base-specific interaction with the third nucleobase is compensated by newly introduced non–base-specific interactions, thereby enabling the NG PAM recognition. We showed that SpCas9-NG induces indels at endogenous target sites bearing NG PAMs in human cells. Furthermore, we found that the fusion of SpCas9-NG and the activation-induced cytidine deaminase (AID) mediates the C-to-T conversion at target sites with NG PAMs in human cells.

scholarly journals Activation-Induced Cytidine Deaminase-Initiated Off-Target DNA Breaks Are Detected and Resolved during S Phase

2012 ◽  
Vol 189 (5) ◽  
pp. 2374-2382 ◽  
Author(s):  
Muneer G. Hasham ◽  
Kathy J. Snow ◽  
Nina M. Donghia ◽  
Jane A. Branca ◽  
Mark D. Lessard ◽  
...  
Keyword(s):  
Cytidine Deaminase ◽  
S Phase ◽  
Dna Breaks ◽  
Target Dna ◽  
Activation Induced Cytidine Deaminase

scholarly journals An optimized base editor with efficient C-to-T base editing in zebrafish

BMC Biology ◽  
2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Yu Zhao ◽  
Dantong Shang ◽  
Ruhong Ying ◽  
Hanhua Cheng ◽  
Rongjia Zhou
Keyword(s):  
Gene Editing ◽  
Cytidine Deaminase ◽  
Model Organism ◽  
Model Organisms ◽  
Base Substitution ◽  
Base Editing ◽  
Codon Preference ◽  
Promising Tool ◽  
Human Genes ◽  
Target Sites

Abstract Background Zebrafish is a model organism widely used for the understanding of gene function, including the fundamental basis of human disease, enabled by the presence in its genome of a high number of orthologs to human genes. CRISPR/Cas9 and next-generation gene-editing techniques using cytidine deaminase fused with Cas9 nickase provide fast and efficient tools able to induce sequence-specific single base mutations in various organisms and have also been used to generate genetically modified zebrafish for modeling pathogenic mutations. However, the editing efficiency in zebrafish of currently available base editors is lower than other model organisms, frequently inducing indel formation, which limits the applicability of these tools and calls for the search of more accurate and efficient editors. Results Here, we generated a new base editor (zAncBE4max) with a length of 5560 bp following a strategy based on the optimization of codon preference in zebrafish. Our new editor effectively created C-to-T base substitution while maintaining a high product purity at multiple target sites. Moreover, zAncBE4max successfully generated the Twist2 p.E78K mutation in zebrafish, recapitulating pathological features of human ablepharon macrostomia syndrome (AMS). Conclusions Overall, the zAncBE4max system provides a promising tool to perform efficient base editing in zebrafish and enhances its capacity to precisely model human diseases.

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.

scholarly journals Improving the specificity of adenine base editor using high-fidelity Cas9

2019 ◽  
Author(s):  
Ruisha Hong ◽  
Sidi Ma ◽  
Feng Wang
Keyword(s):  
Gene Therapy ◽  
Streptococcus Pyogenes ◽  
Genomic Dna ◽  
High Fidelity ◽  
Wild Type ◽  
Sequencing Data ◽  
Deep Sequencing Data ◽  
Target Sites ◽  
Target Activity ◽  
Adenine Base

ABSTRACTAdenine base editor (ABE) mediates the conversion of A to G in genomic DNA. In human, approximately 47.8% of known pathogenic SNPs can be corrected by A to G conversion, indicating that ABE have tremendous potential in gene therapy. However, the off-target activity of ABE limits its clinical application. ABE off-target activity in DNA is depended on the bonding of Streptococcus pyogenes Cas9 (SpCas9) on off-target sites [1, 2]. Therefore, using high-fidelity Cas9 should be able to improve the specificity of ABE. Based on this, we replaced the wild-type SpCas9 in ABE7.10 with four high-fidelity Cas9s to improve its specificity. The analysis of target deep sequencing data demonstrate that the specificity of e-ABE is substantially improved compared to conventional ABE7.10 in four test sites. But the broad editing window of ABE hampers its application, ABE needs to be optimized to get variants with narrow editing window.

scholarly journals The cell cycle restricts activation-induced cytidine deaminase activity to early G1

2016 ◽  
Vol 214 (1) ◽  
pp. 49-58 ◽  
Author(s):  
Qiao Wang ◽  
Kyong-Rim Kieffer-Kwon ◽  
Thiago Y. Oliveira ◽  
Christian T. Mayer ◽  
Kaihui Yao ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Nuclear Membrane ◽  
Nuclear Dna ◽  
Time Window ◽  
Cytidine Deaminase ◽  
Dna Lesions ◽  
Cytidine Deaminase Activity ◽  
Activation Induced Cytidine Deaminase ◽  
Target Sites

Activation-induced cytidine deaminase (AID) converts cytosine into uracil to initiate somatic hypermutation (SHM) and class switch recombination (CSR) of antibody genes. In addition, this enzyme produces DNA lesions at off-target sites that lead to mutations and chromosome translocations. However, AID is mostly cytoplasmic, and how and exactly when it accesses nuclear DNA remains enigmatic. Here, we show that AID is transiently in spatial contact with genomic DNA from the time the nuclear membrane breaks down in prometaphase until early G1, when it is actively exported into the cytoplasm. Consistent with this observation, the immunoglobulin (Igh) gene deamination as measured by uracil accumulation occurs primarily in early G1 after chromosomes decondense. Altering the timing of cell cycle–regulated AID nuclear residence increases DNA damage at off-target sites. Thus, the cell cycle–controlled breakdown and reassembly of the nuclear membrane and the restoration of transcription after mitosis constitute an essential time window for AID-induced deamination, and provide a novel DNA damage mechanism restricted to early G1.

scholarly journals Multiple gene substitution by Target-AID base-editing technology in tomato

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Johan Hunziker ◽  
Keiji Nishida ◽  
Akihiko Kondo ◽  
Sanae Kishimoto ◽  
Tohru Ariizumi ◽  
...  
Keyword(s):  
Target Gene ◽  
Cytidine Deaminase ◽  
Nucleotide Substitutions ◽  
Efficient Tool ◽  
Multiple Gene ◽  
Base Editing ◽  
Carotenoid Accumulation ◽  
Multiple Genes ◽  
Single Target ◽  
Activation Induced Cytidine Deaminase

AbstractThe use of Target activation-induced cytidine deaminase (Target-AID) base-editing technology with the CRISPR-Cas 9 system fused with activation-induced cytidine deaminase (AID) resulted in the substitution of a cytidine with a thymine. In previous experiments focusing on a single target gene, this system has been reported to work in several plant species, including tomato (Solanum lycopersicum L.). In this research, we used Target-AID technology to target multiple genes related to carotenoid accumulation in tomato. We selected 3 genes, SlDDB1, SlDET1 and SlCYC-B, for their roles in carotenoid accumulation. Among 12 edited T0 lines, we obtained 10 independent T0 lines carrying nucleotide substitutions in the three targeted genes, with several allelic versions for each targeted gene. The two edited lines showed significant differences in carotenoid accumulation. These results demonstrate that Target-AID technology is a highly efficient tool for targeting multiple genes with several allelic versions.

scholarly journals Enhancers Improve the AID-Induced Hypermutation in Episomal Vector for Antibody Affinity Maturation in Mammalian Cell Display

Antibodies ◽  
2018 ◽  
Vol 7 (4) ◽  
pp. 42
Author(s):  
Chuan Chen ◽  
Jie Wang ◽  
Yun Zhao ◽  
Shaopeng Chen ◽  
Zhishang Hu ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Somatic Hypermutation ◽  
Cytidine Deaminase ◽  
High Mobility ◽  
Affinity Maturation ◽  
Antibody Affinity ◽  
H1299 Cell ◽  
Episomal Vector ◽  
Activation Induced Cytidine Deaminase

The induction of somatic hypermutation (SHM) in various cell lines by activation-induced cytidine deaminase (AID) has been used in protein-directed selection, especially in antibody affinity maturation. Several antibody affinity maturation systems based on mammalian cells have been developed in recent years, i.e., 293T, H1299, Raji and CHO cells. However, the efficiency of in vitro AID-induced hypermutation is low, restricting the application of such systems. In this study, we examined the role of Ig and Ek enhancers in enhancing SHM in the episomal vector pCEP4 that expresses an anti-high mobility group box 1 (HMGB1) full-length antibody. The plasmid containing the two enhancers exhibited two-fold improvement of mutation rate over pCEP4 in an AID expression H1299 cell line (H1299-AID). With the engineered episomal vector, we improved the affinity of this antibody in H1299-AID cells by 20-fold.

scholarly journals CRISPR adenine and cytosine base editors with reduced RNA off-target activities

2019 ◽  
Author(s):  
Julian Grünewald ◽  
Ronghao Zhou ◽  
Sowmya Iyer ◽  
Caleb A. Lareau ◽  
Sara P. Garcia ◽  
...  
Keyword(s):  
Rna Editing ◽  
Cytidine Deaminase ◽  
Single Nucleotide ◽  
Cytidine Deaminases ◽  
Guide Rna ◽  
Adenosine Deaminases ◽  
Dna Base ◽  
Target Dna ◽  
Activation Induced Cytidine Deaminase ◽  
Target Rna

AbstractCRISPR-guided DNA base editors enable the efficient installation of targeted single-nucleotide changes. Cytosine or adenine base editors (CBEs or ABEs), which are fusions of cytidine or adenosine deaminases to CRISPR-Cas nickases, can efficiently induce DNA C-to-T or A-to-G alterations in DNA, respectively1-4. We recently demonstrated that both the widely used CBE BE3 (harboring a rat APOBEC1 cytidine deaminase) and the optimized ABEmax editor can induce tens of thousands of guide RNA-independent, transcriptome-wide RNA base edits in human cells with high efficiencies5. In addition, we showed the feasibility of creating SElective Curbing of Unwanted RNA Editing (SECURE)-BE3 variants that exhibit substantially reduced unwanted RNA editing activities while retaining robust and more precise on-target DNA editing5. Here we describe structure-guided engineering of SECURE-ABE variants that not only possess reduced off-target RNA editing with comparable on-target DNA activities but are also the smallest Streptococcus pyogenes Cas9 (SpCas9) base editors described to date. In addition, we tested CBEs composed of cytidine deaminases other than APOBEC1 and found that human APOBEC3A (hA3A) cytidine deaminase CBE induces substantial transcriptome-wide RNA base edits with high efficiencies. By contrast, a previously described “enhanced” A3A (eA3A) cytidine deaminase CBE or a human activation-induced cytidine deaminase (hAID) CBE induce substantially reduced or near background levels of RNA edits. In sum, our work describes broadly useful SECURE-ABE and -CBE base editors and reinforces the importance of minimizing RNA editing activities of DNA base editors for research and therapeutic applications.

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