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Iyer, Sowmya

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Iyer

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Sowmya

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Iyer, Sowmya
Author's Publications: search.filters.isAuthorOfPublication.578b32fc-83a9-4df9-b905-1d715709f655

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    Transcriptome-Wide Off-Target RNA Editing Induced by CRISPR-Guided DNA Base Editors
    (Springer Science and Business Media LLC, 2019-04-17) Grünewald, Julian; Zhou, Ronghao; Garcia, Sara; Iyer, Sowmya; Lareau, Caleb; Aryee, Martin; Joung, Keith
    CRISPR-Cas base editor technology enables targeted nucleotide alterations and is being rapidly deployed for research and potential therapeutic applications. The most widely used base editors induce DNA cytosine (C) deamination with rat APOBEC1 (rAPOBEC1) enzyme, which is targeted by a linked Cas protein-guide RNA (gRNA) complex. Previous studies of cytosine base editor (CBE) specificity have identified off-target DNA edits in human cells. Here we show that a CBE with rAPOBEC1 can cause extensive transcriptome-wide RNA cytosine deamination in human cells, inducing tens of thousands of C-to-uracil (U) edits with frequencies ranging from 0.07% to 100% in 38% - 58% of expressed genes. CBE-induced RNA edits occur in both protein-coding and non-protein-coding sequences and generate missense, nonsense, splice site, 5’ UTR, and 3’ UTR mutations. We engineered two CBE variants bearing rAPOBEC1 mutations that substantially decrease the numbers of RNA edits (reductions of >390-fold and >3,800-fold) in human cells. These variants also showed more precise on-target DNA editing and, with the majority of gRNAs tested, editing efficiencies comparable to those observed with wild-type CBE. Finally, we show that recently described adenine base editors (ABEs) can also induce transcriptome-wide RNA edits. These results have important implications for the research and therapeutic uses of base editors, illustrate the feasibility of engineering improved variants with reduced RNA editing activities, and suggest the need to more fully define and characterize the RNA off-target effects of deaminase enzymes in base editor platforms.
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    Augmenting and Directing Robust, Long-Range CRISPR-Mediated Activation in Human Cells
    (Springer Science and Business Media LLC, 2021-08-05) Tak, Y. Esther; Horng, Joy E.; Perry, Nicholas T.; Schultz, Hayley; Iyer, Sowmya; Yao, Qiuming; Zou, Luli S.; Aryee, Martin; Pinello, Luca; Joung, Keith
    Epigenetic editing is an emerging technology that uses artificial transcription factors (aTFs) to regulate expression of a target gene. Although human genes can be robustly upregulated by targeting aTFs to promoters, the activation induced by targeting aTFs to distal transcriptional enhancers is substantially less robust and consistent. Here we show that long-range activation using CRISPR-based aTFs in human cells can be made more efficient and reliable by concurrently targeting an aTF to the target gene promoter. We used this strategy to direct target gene choice for enhancers capable of regulating more than one promoter and to achieve allele-selective activation of human genes by targeting aTFs to SNPs embedded in distally located sequences. Our results broaden the potential applications of the epigenetic editing toolbox for research and therapeutics.
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    CRISPR DNA Base Editors With Reduced RNA Off-Target and Self-Editing Activities
    (Springer Science and Business Media LLC, 2019-09) Grünewald, Julian; Zhou, Ronghao; Iyer, Sowmya; Lareau, Caleb; Garcia, Sara; Aryee, Martin; Joung, Keith
    Cytosine or adenine base editors (CBEs or ABEs) can introduce specific DNA C-to-T or A-to-G alterations1,2,3,4. 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.