Evaluation of off‐target effects of gapmer antisense oligonucleotides using human cells

Cytosine base editors (CBEs) enable efficient cytidine-to-thymidine (C-to-T) substitutions at targeted loci without double-stranded breaks. However, current CBEs edit all Cs within their activity windows, generating undesired bystander mutations. In the most challenging circumstance, when a bystander C is adjacent to the targeted C, existing base editors fail to discriminate them and edit both Cs. To improve the precision of CBE, we identified and engineered the human APOBEC3G (A3G) deaminase; when fused to the Cas9 nickase, the resulting A3G-BEs exhibit selective editing of the second C in the 5′-CC-3′ motif in human cells. Our A3G-BEs could install a single disease-associated C-to-T substitution with high precision. The percentage of perfectly modified alleles is more than 6000-fold for disease correction and more than 600-fold for disease modeling compared with BE4max. On the basis of the two-cell embryo injection method and RNA sequencing analysis, our A3G-BEs showed minimum genome- and transcriptome-wide off-target effects, achieving high targeting fidelity.

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Identifying off-target effects and hidden phenotypes of drugs in human cells

Nature Chemical Biology ◽

10.1038/nchembio790 ◽

2006 ◽

Vol 2 (6) ◽

pp. 329-337 ◽

Cited By ~ 224

Author(s):

Marnie L MacDonald ◽

Jane Lamerdin ◽

Stephen Owens ◽

Brigitte H Keon ◽

Graham K Bilter ◽

...

Keyword(s):

Human Cells ◽

Target Effects

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Specificity of RNAi, LNA and CRISPRi as loss-of-function methods in transcriptional analysis

10.1101/234930 ◽

2017 ◽

Cited By ~ 1

Author(s):

Lovorka Stojic ◽

Aaron Lun ◽

Jasmin Mangei ◽

Patrice Mascalchi ◽

Valentina Quarantotti ◽

...

Keyword(s):

Antisense Oligonucleotides ◽

Noncoding Rna ◽

Specific Activity ◽

Transcriptional Analysis ◽

Clonal Variation ◽

Loss Of Function ◽

Negative Controls ◽

Whole Transcriptome ◽

Target Effects

ABSTRACTLoss-of-function (LOF) methods, such as RNA interference (RNAi), antisense oligonucleotides or CRISPR-based genome editing, provide unparalleled power for studying the biological function of genes of interest. When coupled with transcriptomic analyses, LOF methods allow researchers to dissect networks of transcriptional regulation. However, a major concern is nonspecific targeting, which involves depletion of transcripts other than those intended. The off-target effects of each of these common LOF methods have yet to be compared at the whole-transcriptome level. Here, we systematically and experimentally compared non-specific activity of RNAi, antisense oligonucleotides and CRISPR interference (CRISPRi). All three methods yielded non-negligible offtarget effects in gene expression, with CRISPRi exhibiting clonal variation in the transcriptional profile. As an illustrative example, we evaluated the performance of each method for deciphering the role of a long noncoding RNA (lncRNA) with unknown function. Although all LOF methods reduced expression of the candidate lncRNA, each method yielded different sets of differentially expressed genes upon knockdown as well as a different cellular phenotype. Therefore, to definitively confirm the functional role of a transcriptional regulator, we recommend the simultaneous use of at least two different LOF methods and the inclusion of multiple, specifically designed negative controls.

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Design and delivery of antisense oligonucleotides to block microRNA function in cultured Drosophila and human cells

Nature Protocols ◽

10.1038/nprot.2008.145 ◽

2008 ◽

Vol 3 (10) ◽

pp. 1537-1549 ◽

Cited By ~ 71

Author(s):

Michael D Horwich ◽

Phillip D Zamore

Keyword(s):

Antisense Oligonucleotides ◽

Human Cells ◽

Microrna Function

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No observable guide-RNA-independent off-target mutation induced by prime editor

10.1101/2021.04.09.439109 ◽

2021 ◽

Author(s):

Runze Gao ◽

Zhi-Can Fu ◽

Xiangyang Li ◽

Ying Wang ◽

Jia Wei ◽

...

Keyword(s):

Transcriptome Sequencing ◽

High Specificity ◽

Human Cells ◽

Whole Genome ◽

Guide Rna ◽

Genome Wide ◽

Target Sites ◽

Whole Transcriptome Sequencing ◽

Whole Transcriptome ◽

Target Effects

Prime editor (PE) has been recently developed to induce efficient and precise on-target editing, whereas its guide RNA (gRNA)-independent off-target effects remain unknown. Here, we used whole-genome and whole-transcriptome sequencing to determine gRNA-independent off-target mutations in cells expanded from single colonies, in which PE generated precise editing at on-target sites. We found that PE triggered no observable gRNA-independent off-target mutation genome-wide or transcriptome-wide in transfected human cells, highlighting its high specificity.

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Highly efficient and specific genome editing in human cells with paired CRISPR-Cas9 nickase ribonucleoproteins

10.1101/766493 ◽

2019 ◽

Author(s):

Jacob Lamberth ◽

Laura Daley ◽

Pachai Natarajan ◽

Stanislav Khoruzhenko ◽

Nurit Becker ◽

...

Keyword(s):

Cell Culture ◽

Genome Editing ◽

Plasmid Dna ◽

High Efficiency ◽

High Specificity ◽

Human Cells ◽

Dna And Rna ◽

Genome Modification ◽

Delivery Technology ◽

Target Effects

ABSTRACTCRISPR technology has opened up many diverse genome editing possibilities in human somatic cells, but has been limited in the therapeutic realm by both potential off-target effects and low genome modification efficiencies. Recent advancements to combat these limitations include delivering Cas9 nucleases directly to cells as highly purified ribonucleoproteins (RNPs) instead of the conventional plasmid DNA and RNA-based approaches. Here, we extend RNP-based delivery in cell culture to a less characterized CRISPR format which implements paired Cas9 nickases. The use of paired nickase Cas9 RNP system, combined with a GMP-compliant non-viral delivery technology, enables editing in human cells with high specificity and high efficiency, a development that opens up the technology for further exploration into a more therapeutic role.

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Splice-switching efficiency and specificity for oligonucleotides with locked nucleic acid monomers

Biochemical Journal ◽

10.1042/bj20080013 ◽

2008 ◽

Vol 412 (2) ◽

pp. 307-313 ◽

Cited By ~ 24

Author(s):

Peter Guterstam ◽

Maria Lindgren ◽

Henrik Johansson ◽

Ulf Tedebark ◽

Jesper Wengel ◽

...

Keyword(s):

Nucleic Acid ◽

Antisense Oligonucleotides ◽

High Efficiency ◽

Locked Nucleic Acid ◽

Switching Activity ◽

Mismatch Discrimination ◽

Therapeutic Platform ◽

Splicing Patterns ◽

Switching Efficiency ◽

Target Effects

The use of antisense oligonucleotides to modulate splicing patterns has gained increasing attention as a therapeutic platform and, hence, the mechanisms of splice-switching oligonucleotides are of interest. Cells expressing luciferase pre-mRNA interrupted by an aberrantly spliced β-globin intron, HeLa pLuc705, were used to monitor the splice-switching activity of modified oligonucleotides by detection of the expression of functional luciferase. It was observed that phosphorothioate 2′-O-methyl RNA oligonucleotides containing locked nucleic acid monomers provide outstanding splice-switching activity. However, similar oligonucleotides with several mismatches do not impede splice-switching activity which indicates a risk for off-target effects. The splice-switching activity is abolished when mismatches are introduced at several positions with locked nucleic acid monomers suggesting that it is the locked nucleic acid monomers that give rise to low mismatch discrimination to target pre-mRNA. The results highlight the importance of rational sequence design to allow for high efficiency with simultaneous high mismatch discrimination for splice-switching oligonucleotides and suggest that splice-switching activity is tunable by utilizing locked nucleic acid monomers.

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Using RNA-seq to Assess Off-Target Effects of Antisense Oligonucleotides in Human Cell Lines

Molecular Diagnosis & Therapy ◽

10.1007/s40291-020-00504-4 ◽

2020 ◽

Author(s):

Sven Michel ◽

Ksenija Schirduan ◽

Yimin Shen ◽

Richard Klar ◽

Jörg Tost ◽

...

Keyword(s):

Cell Lines ◽

Human Cell ◽

Antisense Oligonucleotides ◽

Human Cell Lines ◽

Rna Seq ◽

Target Effects

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Hybridization-mediated off-target effects of splice-switching antisense oligonucleotides

Nucleic Acids Research ◽

10.1093/nar/gkz1132 ◽

2019 ◽

Vol 48 (2) ◽

pp. 802-816 ◽

Cited By ~ 6

Author(s):

Juergen Scharner ◽

Wai Kit Ma ◽

Qian Zhang ◽

Kuan-Ting Lin ◽

Frank Rigo ◽

...

Keyword(s):

Antisense Oligonucleotides ◽

Genetic Diseases ◽

Therapeutic Modality ◽

Target Sequence ◽

Exon Definition ◽

Sequence Complementarity ◽

In Vitro Effects ◽

Target Activity ◽

Target Effects

Abstract Splice-switching antisense oligonucleotides (ASOs), which bind specific RNA-target sequences and modulate pre-mRNA splicing by sterically blocking the binding of splicing factors to the pre-mRNA, are a promising therapeutic modality to treat a range of genetic diseases. ASOs are typically 15–25 nt long and considered to be highly specific towards their intended target sequence, typically elements that control exon definition and/or splice-site recognition. However, whether or not splice-modulating ASOs also induce hybridization-dependent mis-splicing of unintended targets has not been systematically studied. Here, we tested the in vitro effects of splice-modulating ASOs on 108 potential off-targets predicted on the basis of sequence complementarity, and identified 17 mis-splicing events for one of the ASOs tested. Based on analysis of data from two overlapping ASO sequences, we conclude that off-target effects are difficult to predict, and the choice of ASO chemistry influences the extent of off-target activity. The off-target events caused by the uniformly modified ASOs tested in this study were significantly reduced with mixed-chemistry ASOs of the same sequence. Furthermore, using shorter ASOs, combining two ASOs, and delivering ASOs by free uptake also reduced off-target activity. Finally, ASOs with strategically placed mismatches can be used to reduce unwanted off-target splicing events.

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P-glycoprotein–actin association through ERM family proteins: a role in P-glycoprotein function in human cells of lymphoid origin

Blood ◽

10.1182/blood.v99.2.641 ◽

2002 ◽

Vol 99 (2) ◽

pp. 641-648 ◽

Cited By ~ 101

Author(s):

Francesca Luciani ◽

Agnese Molinari ◽

Francesco Lozupone ◽

Annarica Calcabrini ◽

Luana Lugini ◽

...

Keyword(s):

Multidrug Resistance ◽

Actin Cytoskeleton ◽

Antisense Oligonucleotides ◽

Transmembrane Protein ◽

Human Cells ◽

Lymphoid Cells ◽

Ion Pump ◽

Resistant Variant ◽

Resistance Phenotype ◽

P Glycoprotein

Abstract P-glycoprotein is a 170-kd glycosylated transmembrane protein, expressed in a variety of human cells and belonging to the adenosine triphosphate–binding cassette transporter family, whose membrane expression is functionally associated with the multidrug resistance phenotype. However, the mechanisms underlying the regulation of P-glycoprotein functions remain unclear. On the basis of some evidence suggesting P-glycoprotein–actin cytoskeleton interaction, this study investigated the association of P-glycoprotein with ezrin, radixin, and moesin, a class of proteins that cross-link actin filaments with plasma membrane in a human cell line of lymphoid origin and that have been shown to link other ion-pump–related proteins. To this purpose, a multidrug-resistant variant of CCRF-CEM cells (CEM-VBL100) was used as a model to investigate the following: (1) the cellular localizations of P-glycoprotein and ezrin, radixin, and moesin and their molecular associations; and (2) the effects of ezrin, radixin, and moesin antisense oligonucleotides on multidrug resistance and P-glycoprotein function. The results showed that: (1) P-glycoprotein colocalized and coimmunoprecipitated with ezrin, radixin, and moesin; and (2) treatment with antisense oligonucleotides for ezrin, radixin, and moesin restored drug susceptibility consistently with inhibition of both drug efflux and actin–P-glycoprotein association and induction of cellular redistribution of P-glycoprotein. These data suggest that P-glycoprotein association with the actin cytoskeleton through ezrin, radixin, and moesin is key in conferring to human lymphoid cells a multidrug resistance phenotype. Strategies aimed at inhibiting P-glycoprotein–actin association may be helpful in increasing the efficiency of both antitumor and antiviral therapies.

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