Browse
Submit Data
Databases
API
Help

Dataset Information

17 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

A general theoretical framework to design base editors with reduced bystander effects

ABSTRACT: Base editors (BEs) hold great potential for medical applications of gene therapy. However, high precision base editing requires BEs that can discriminate between the target base and multiple bystander bases within a narrow active window (4 – 10 nucleotides). Here, to assist in the design of these optimized editors, we propose a discrete-state stochastic approach to build an analytical model that explicitly evaluates the probabilities of editing the target base and bystanders. Combined with all-atom molecular dynamic simulations, our model reproduces the experimental data of A3A-BE3 and its variants for targeting the “TC” motif and bystander editing. Analyzing this approach, we propose several general principles that can guide the design of BEs with a reduced bystander effect. These principles are then applied to design a series of point mutations at T218 position of A3G-BEs to further reduce its bystander editing. We verify experimentally that the new mutations provide different levels of stringency on reducing the bystander editing at different genomic loci, which is consistent with our theoretical model. Thus, our study provides a computational-aided platform to assist in the scientifically-based design of BEs with reduced bystander effects. Base editors can edit target nucleotides, and identical ones that are within the editing window. Here the authors build an analytical model to propose general principles of editor design to reduce bystander effects.

SUBMITTER: Wang Q

PROVIDER: S-EPMC8586357 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

CRISPR DNA base editors with reduced RNA off-target and self-editing activities.

Project description:Cytosine or adenine base editors (CBEs or ABEs) can introduce specific DNA C-to-T or A-to-G alterations1-4. However, we recently demonstrated that they can also induce transcriptome-wide guide-RNA-independent editing of RNA bases5, and created selective curbing of unwanted RNA editing (SECURE)-BE3 variants that have reduced unwanted RNA-editing activity5. Here we describe structure-guided engineering of SECURE-ABE variants with reduced off-target RNA-editing activity and comparable on-target DNA-editing activity that are also among the smallest Streptococcus pyogenes Cas9 base editors described to date. We also tested CBEs with cytidine deaminases other than APOBEC1 and found that the human APOBEC3A-based CBE induces substantial editing of RNA bases, whereas an enhanced APOBEC3A-based CBE6, human activation-induced cytidine deaminase-based CBE7, and the Petromyzon marinus cytidine deaminase-based CBE Target-AID4 induce less editing of RNA. Finally, we found that CBEs and ABEs that exhibit RNA off-target editing activity can also self-edit their own transcripts, thereby leading to heterogeneity in base-editor coding sequences.

| S-EPMC6730565 | biostudies-literature

AcrIIA5 Suppresses Base Editors and Reduces Their Off-Target Effects.

Project description:The CRISPR/nCas9-based cytosine base editors (CBEs) and adenine base editors (ABEs) are capable of catalyzing C•G to T•A or A•T to G•C conversions, respectively, and have become new, powerful tools for achieving precise genetic changes in a wide range of organisms. These base editors hold great promise for correcting pathogenic mutations and for being used for therapeutic applications. However, the recognition of cognate DNA sequences near their target sites can cause severe off-target effects that greatly limit their clinical applications, and this is an urgent problem that needs to be resolved for base editing systems. The recently discovered phage-derived proteins, anti-CRISPRs, which can suppress the natural CRISPR nuclease activity, may be able to ameliorate the off-target effects of base editing systems. Here, we confirm for the first time that AcrIIA2, AcrIIA4, and AcrIIA5 efficiently inhibit base editing systems in human cells. In particular, AcrIIA5 has a significant inhibitory effect on all base editing variant systems tested in our study. We further show that the off-target effects of BE3 and ABE7.10 were significantly reduced in AcrIIA5 treated cells. This study suggests that AcrIIA5 should be widely used for the precise control of base editing and to thoroughly "shut off" nuclease activity of both CBE and ABE systems.

| S-EPMC7463901 | biostudies-literature

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

Project description:This SuperSeries is composed of the SubSeries listed below.

2019-07-11 | GSE129894 | GEO

CRISPR adenine and cytosine base editors with reduced RNA off-target activities [ABE]

Project description:CRISPR-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, respectively. 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 efficiencies. 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 editing. 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.

2019-07-11 | GSE129889 | GEO

CRISPR adenine and cytosine base editors with reduced RNA off-target activities [CBE]

2019-07-11 | GSE129893 | GEO

A Cas-embedding strategy for minimizing off-target effects of DNA base editors.

Project description:DNA base editors, typically comprising editing enzymes fused to the N-terminus of nCas9, display off-target effects on DNA and/or RNA, which have remained an obstacle to their clinical applications. Off-target edits are typically countered via rationally designed point mutations, but the approach is tedious and not always effective. Here, we report that the off-target effects of both A > G and C > T editors can be dramatically reduced without compromising the on-target editing simply by inserting the editing enzymes into the middle of nCas9 at tolerant sites identified using a transposon-based genetic screen. Furthermore, employing this Cas-embedding strategy, we have created a highly specific editor capable of efficient C > T editing at methylated and GC-rich sequences.

| S-EPMC7695861 | biostudies-literature

Internally inlaid SaCas9 base editors enable window specific base editing.

Project description:Rationale: Base editors composed of catalytic defective Cas9 and cytosine or adenosine deaminase are powerful tools to convert bases in a genome. However, the fixed and narrow editing window of current base editors has impeded their utility. To increase the scope and diversify the editing patterns is quite necessary. Methods and Results: We designed a subset of base editors derived from SaCas9 in which deaminase was inlaid into various locations of the SaCas9 protein. The resulting base editors were characterized with multiple genomic sites and were found to have distinct editing features to the N-terminal SaCas9 CBE (Sa-CBE-N). Among them, Sa-CBE-693, in which a cytosine deaminase was inserted between amino acids 693 and 694, showed an increased editing efficiency and a significantly expanded editing window ranging from bases 2-18. This feature enhanced the editing efficiency of BCL11A enhancer that contains multiple consensus bases in a 15-bp fragment. Another variant, Sa-CBE-125, displayed backward-shifted editing window, which we showed was particularly powerful in editing cytosines that were accompanied with unintended bystander cytosines at their 5' side. Additionally, these editors showed reduced Cas9 independent DNA off-target editing compared with Sa-CBE-N. Conclusion: Our inlaid base editors improved the targeting scope and diversified the editing pattern.

| S-EPMC9254239 | biostudies-literature

Simplified adenine base editors improve adenine base editing efficiency in rice.

Project description:Adenine base editors (ABEs) have been exploited to introduce targeted adenine (A) to guanine (G) base conversions in various plant genomes, including rice, wheat and Arabidopsis. However, the ABEs reported thus far are all quite inefficient at many target sites in rice, which hampers their applications in plant genome engineering and crop breeding. Here, we show that unlike in the mammalian system, a simplified base editor ABE-P1S (Adenine Base Editor-Plant version 1 Simplified) containing the ecTadA*7.10-nSpCas9 (D10A) fusion has much higher editing efficiency in rice compared to the widely used ABE-P1 consisting of the ecTadA-ecTadA*7.10-nSpCas9 (D10A) fusion. We found that the protein expression level of ABE-P1S is higher than that of ABE-P1 in rice calli and protoplasts, which may explain the higher editing efficiency of ABE-P1S in different rice varieties. Moreover, we demonstrate that the ecTadA*7.10-nCas9 fusion can be used to improve the editing efficiency of other ABEs containing SaCas9 or the engineered SaKKH-Cas9 variant. These more efficient ABEs will help advance trait improvements in rice and other crops.

| S-EPMC7004905 | biostudies-literature

Computer simulations explain mutation-induced effects on the DNA editing by adenine base editors.

Project description:Adenine base editors, which were developed by engineering a transfer RNA adenosine deaminase enzyme (TadA) into a DNA editing enzyme (TadA*), enable precise modification of A:T to G⋮C base pairs. Here, we use molecular dynamics simulations to uncover the structural and functional roles played by the initial mutations in the onset of the DNA editing activity by TadA*. Atomistic insights reveal that early mutations lead to intricate conformational changes in the structure of TadA*. In particular, the first mutation, Asp108Asn, induces an enhancement in the binding affinity of TadA to DNA. In silico and in vivo reversion analyses verify the importance of this single mutation in imparting functional promiscuity to TadA* and demonstrate that TadA* performs DNA base editing as a monomer rather than a dimer.

| S-EPMC7056309 | biostudies-literature

Rational Design of Azastatin as a Potential ADC Payload with Reduced Bystander Killing.

Project description:Auristatins are a class of ultrapotent microtubule inhibitors, whose growing clinical popularity in oncology is based upon their use as payloads in antibody-drug conjugates (ADCs). The most widely utilized auristatin, MMAE, has however been shown to cause apoptosis in non-pathological cells proximal to the tumour ("bystander killing"). Herein, we introduce azastatins, a new class of auristatin derivatives encompassing a side chain amine for antibody conjugation. The synthesis of Cbz-azastatin methyl ester, which included the C2-elongation and diastereoselective reduction of two proteinogenic amino acids as key transformations, was accomplished in 22 steps and 0.76?% overall yield. While Cbz-protected azastatin methyl ester (0.13-3.0?nM) inhibited proliferation more potently than MMAE (0.47-6.5?nM), removal of the Cbz-group yielded dramatically increased IC50 -values (9.8-170?nM). We attribute the reduced apparent cytotoxicity of the deprotected azastatin methyl esters to a lack of membrane permeability. These results clearly establish the azastatins as a novel class of cytotoxic payloads ideally suited for use in next-generation ADC development.

| S-EPMC7756782 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data