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.

scholarly journals Cytosine and adenine deaminase base-editors induce broad and nonspecific changes in gene expression and splicing

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Jiao Fan ◽  
Yige Ding ◽  
Chao Ren ◽  
Ziguo Song ◽  
Jie Yuan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Double Strand Breaks ◽  
Great Promise ◽  
Single Nucleotide ◽  
Strand Breaks ◽  
Dna Base ◽  
Target Dna ◽  
Single Nucleotide Variations ◽  
Adenine Deaminase ◽  
Target Rna

AbstractCytosine or adenine base editors (CBEs or ABEs) hold great promise in therapeutic applications because they enable the precise conversion of targeted base changes without generating of double-strand breaks. However, both CBEs and ABEs induce substantial off-target DNA editing, and extensive off-target RNA single nucleotide variations in transfected cells. Therefore, the potential effects of deaminases induced by DNA base editors are of great importance for their clinical applicability. Here, the transcriptome-wide deaminase effects on gene expression and splicing is examined. Differentially expressed genes (DEGs) and differential alternative splicing (DAS) events, induced by base editors, are identified. Both CBEs and ABEs generated thousands of DEGs and hundreds of DAS events. For engineered CBEs or ABEs, base editor-induced variants had little effect on the elimination of DEGs and DAS events. Interestingly, more DEGs and DAS events are observed as a result of over expressions of cytosine and adenine deaminases. This study reveals a previously overlooked aspect of deaminase effects in transcriptome-wide gene expression and splicing, and underscores the need to fully characterize such effects of deaminase enzymes in base editor platforms.

scholarly journals Specific Expression of Activation-induced Cytidine Deaminase (AID), a Novel Member of the RNA-editing Deaminase Family in Germinal Center B Cells

1999 ◽  
Vol 274 (26) ◽  
pp. 18470-18476 ◽  
Author(s):  
Masamichi Muramatsu ◽  
V. S. Sankaranand ◽  
Shrikant Anant ◽  
Manabu Sugai ◽  
Kazuo Kinoshita ◽  
...  
Keyword(s):  
B Cells ◽  
Rna Editing ◽  
Germinal Center ◽  
Cytidine Deaminase ◽  
Specific Expression ◽  
Activation Induced Cytidine Deaminase ◽  
Germinal Center B 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 Precise adenine base editors that exhibit minimized cytosine catalysis

2020 ◽  
Author(s):  
You Kyeong Jeong ◽  
SeokHoon Lee ◽  
Gue-Ho Hwang ◽  
Sung-Ah Hong ◽  
Se-eun Park ◽  
...  
Keyword(s):  
Synergistic Effect ◽  
Adenosine Deaminase ◽  
Rna Editing ◽  
The Other ◽  
Cytosine Deamination ◽  
Base Editing ◽  
Target Dna ◽  
Editing Activity ◽  
Target Rna ◽  
Adenine Base

Abstract Adenine base editors (ABEs) promise specific A-to-G conversions at genomic sites of interest. However, ABEs also induce cytosine deamination at the target DNA site and exhibit transcriptome-wide off-target RNA editing. To alleviate the ABE-mediated cytosine editing activity, here we engineered the commonly-used version of adenosine deaminase, TadA7.10, to contain rationally designed mutations. We ultimately found that ABE7.10 with a D108Q mutation in TadA7.10 exhibited greatly reduced cytosine deamination activity, and conversely, ABE7.10 containing a P48R mutation displayed increased cytosine deamination activity rather than adenine editing. We found that the D108Q mutation also reduces cytosine deamination activity in two recently-developed versions of ABE, ABE8e and ABE8s, and has a synergistic effect with V106W, a key mutation that reduces off-target RNA editing. On the other hand, by incorporating the P48R mutation into ABE7.10, we demonstrated TC-specific base editing tools that enable either TC-to-TT or TC-to-TG conversions, broadening the utility of base editors.

scholarly journals CRISPR-Cas9 bends and twists DNA to read its sequence

2021 ◽  
Author(s):  
Joshua C. Cofsky ◽  
Katarzyna M. Soczek ◽  
Gavin J. Knott ◽  
Eva Nogales ◽  
Jennifer A. Doudna
Keyword(s):  
Genome Editing ◽  
Target Sequence ◽  
Base Flipping ◽  
Base Pairs ◽  
Guide Rna ◽  
Target Capture ◽  
Dna Base ◽  
Protospacer Adjacent Motif ◽  
Target Dna ◽  
Dna Base Pairs

In bacterial defense and genome editing applications, the CRISPR-associated protein Cas9 searches millions of DNA base pairs to locate a 20-nucleotide, guide-RNA-complementary target sequence that abuts a protospacer-adjacent motif (PAM)1. Target capture requires Cas9 to unwind DNA at candidate sequences using an unknown ATP-independent mechanism2,3. Here we show that Cas9 sharply bends and undertwists DNA at each PAM, thereby flipping DNA nucleotides out of the duplex and toward the guide RNA for sequence interrogation. Cryo-electron-microscopy (EM) structures of Cas9:RNA:DNA complexes trapped at different states of the interrogation pathway, together with solution conformational probing, reveal that global protein rearrangement accompanies formation of an unstacked DNA hinge. Bend-induced base flipping explains how Cas9 “reads” snippets of DNA to locate target sites within a vast excess of non-target DNA, a process crucial to both bacterial antiviral immunity and genome editing. This mechanism establishes a physical solution to the problem of complementarity-guided DNA search and shows how interrogation speed and local DNA geometry may influence genome editing efficiency.

scholarly journals DYW domain structures imply an unusual regulation principle in plant organellar RNA editing catalysis

2021 ◽  
Vol 4 (6) ◽  
pp. 510-522
Author(s):  
Mizuki Takenaka ◽  
Sachi Takenaka ◽  
Tatjana Barthel ◽  
Brody Frink ◽  
Sascha Haag ◽  
...  
Keyword(s):  
Rna Editing ◽  
Ground State ◽  
Cytidine Deaminase ◽  
Pentatricopeptide Repeat ◽  
Domain Structures ◽  
Differential Scanning Fluorimetry ◽  
Pentatricopeptide Repeat Proteins ◽  
Cytidine Deamination ◽  
Target Rna

AbstractRNA editosomes selectively deaminate cytidines to uridines in plant organellar transcripts—mostly to restore protein functionality and consequently facilitate mitochondrial and chloroplast function. The RNA editosomal pentatricopeptide repeat proteins serve target RNA recognition, whereas the intensively studied DYW domain elicits catalysis. Here we present structures and functional data of a DYW domain in an inactive ground state and activated. DYW domains harbour a cytidine deaminase fold and a C-terminal DYW motif, with catalytic and structural zinc atoms, respectively. A conserved gating domain within the deaminase fold regulates the active site sterically and mechanistically in a process that we termed gated zinc shutter. Based on the structures, an autoinhibited ground state and its activation are cross-validated by RNA editing assays and differential scanning fluorimetry. We anticipate that, in vivo, the framework of an active plant RNA editosome triggers the release of DYW autoinhibition to ensure a controlled and coordinated cytidine deamination playing a key role in mitochondrial and chloroplast homeostasis.

scholarly journals Class Switch Recombination and Hypermutation Require Activation-Induced Cytidine Deaminase (AID), a Potential RNA Editing Enzyme

Cell ◽  
2000 ◽  
Vol 102 (5) ◽  
pp. 553-563 ◽  
Author(s):  
Masamichi Muramatsu ◽  
Kazuo Kinoshita ◽  
Sidonia Fagarasan ◽  
Shuichi Yamada ◽  
Yoichi Shinkai ◽  
...  
Keyword(s):  
Rna Editing ◽  
Cytidine Deaminase ◽  
Class Switch Recombination ◽  
Class Switch ◽  
Activation Induced Cytidine Deaminase ◽  
Editing Enzyme

scholarly journals Analisis Polimorfisme Nukleotida Tunggal (SNP) Daerah 3’UTR Gen LDLR Penduduk Papua

2019 ◽  
Vol 15 (1) ◽  
pp. 11-20
Author(s):  
Asri Saffanah Pratiwi ◽  
Achmad Taher
Keyword(s):  
Receptor Gene ◽  
Low Density Lipoprotein ◽  
Haplotype Diversity ◽  
Density Lipoprotein ◽  
Polymerase Chain Reaction Method ◽  
Ldlr Gene ◽  
Single Nucleotide ◽  
Cholesterol Levels ◽  
Dna Base ◽  
Target Dna

Single nucleotide polymorphism (SNP) is a single nucleotide difference in the arrangement of DNA base strands that can show genetic variation. The LDLR gene is a low density lipoprotein (LDL-R) receptor gene that functions to regulate cholesterol levels in the blood. The LDLR gene is composed of 18 exons and contains a 3’untranslated region (3’UTR) which plays an important role in regulating gene expression. This study aims to analyze the SNP in an area of 3'UTR LDLR genes from 6 University of Papua students from Papua. The research was carried out by polymerase chain reaction method to multiply the number of target DNA, then sequenced to find out the sequence of nucleotide bases. The results of this study were from 6 individuals, found 2 SNPs at position *52 and *504 with nucleotide diversity (π) of 0.00149. These polymorphisms forms 3 types of haplotypes, namely GG, GA and AA with a haplotype diversity of 0.600 ± 0.215.

scholarly journals CRISPR-Cas9 bends and twists DNA to read its sequence

2021 ◽  
Author(s):  
Jennifer Doudna ◽  
Joshua Cofsky ◽  
Katarzyna Soczek ◽  
Gavin Knott ◽  
Eva Nogales
Keyword(s):  
Genome Editing ◽  
Target Sequence ◽  
Base Flipping ◽  
Base Pairs ◽  
Guide Rna ◽  
Target Capture ◽  
Dna Base ◽  
Protospacer Adjacent Motif ◽  
Target Dna ◽  
Dna Base Pairs

Abstract In bacterial defense and genome editing applications, the CRISPR-associated protein Cas9 searches millions of DNA base pairs to locate a 20-nucleotide, guide-RNA-complementary target sequence that abuts a protospacer-adjacent motif (PAM). Target capture requires Cas9 to unwind DNA at candidate sequences using an unknown ATP-independent mechanism. Here we show that Cas9 sharply bends and undertwists DNA at each PAM, thereby flipping DNA nucleotides out of the duplex and toward the guide RNA for sequence interrogation. Cryo-electron-microscopy (EM) structures of Cas9:RNA:DNA complexes trapped at different states of the interrogation pathway, together with solution conformational probing, reveal that global protein rearrangement accompanies formation of an unstacked DNA hinge. Bend-induced base flipping explains how Cas9 “reads” snippets of DNA to locate target sites within a vast excess of non-target DNA, a process crucial to both bacterial antiviral immunity and genome editing. This mechanism establishes a physical solution to the problem of complementarity-guided DNA search and shows how interrogation speed and local DNA geometry may influence genome editing efficiency.

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