Transition Substitution of Desired Bases in Human Pluripotent Stem Cells with Base Editors: A Step-by-Step Guide

Park, Ju-Chan; Kim, Keun-Tae; Jang, Hyeon-Ki; Cha, Hyuk‐Jin

doi:10.15283/ijsc22171

Cited by 4 publications

(5 citation statements)

References 32 publications

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“…The same amount of siNC and pcDNA 3.0 was added to the cell solution for the control of siUNG and p53DD respectively. The electroporation was performed with NEPA-21 electroporator with 175 V, 2.5 ms of poring pulse as described 20 . Dissociated cells after electroporation were plated into a culture dish with 10 μM of Y-27632.…”

Section: Methodsmentioning

confidence: 99%

“…Since base editors (BEs) and prime editor (PE) technology developed from nickase Cas9 (nCas9) introduce the intended genetic variations without DSBs 12 , 13 , the potential utility of BEs in clinical applications has received heightened interest 14 . Accordingly, BEs 15 – 20 and PE technology 21 have been applied in hPSCs for either disease modeling or mutation correction 22 .…”

Section: Introductionmentioning

confidence: 99%

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Enhancing genome editing in hPSCs through dual inhibition of DNA damage response and repair pathways

Park,

Kim,

Hwang

et al. 2024

Nat Commun

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Precise genome editing is crucial for establishing isogenic human disease models and ex vivo stem cell therapy from the patient-derived hPSCs. Unlike Cas9-mediated knock-in, cytosine base editor and prime editor achieve the desirable gene correction without inducing DNA double strand breaks. However, hPSCs possess highly active DNA repair pathways and are particularly susceptible to p53-dependent cell death. These unique characteristics impede the efficiency of gene editing in hPSCs. Here, we demonstrate that dual inhibition of p53-mediated cell death and distinct activation of the DNA damage repair system upon DNA damage by cytosine base editor or prime editor additively enhanced editing efficiency in hPSCs. The BE4stem system comprised of p53DD, a dominant negative p53, and three UNG inhibitor, engineered to specifically diminish base excision repair, improves cytosine base editor efficiency in hPSCs. Addition of dominant negative MLH1 to inhibit mismatch repair activity and p53DD in the conventional prime editor system also significantly enhances prime editor efficiency in hPSCs. Thus, combined inhibition of the distinct cellular cascades engaged in hPSCs upon gene editing could significantly enhance precise genome editing in these cells.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Enhancing genome editing in hPSCs through dual inhibition of DNA damage response and repair pathways

Park,

Kim,

Hwang

et al. 2024

Nat Commun

Self Cite

View full text Add to dashboard Cite

show abstract

“…Same amount of siNC and pcDNA 3.0 was add to cell solution for the control of siUNG and p53DD respectively. The electroporation was performed with NEPA-21 electroporator with 175 V, 2.5ms of poring pulse as described 20 .…”

Section: Methodsmentioning

confidence: 99%

“…Since base editors (BEs) and prime editor (PE) technology developed from nickase Cas9 (nCas9) introduce the intended genetic variations without DSBs 12,13 , the potential utility of BEs in clinical applications has received heightened interest 14 . Accordingly, BEs [15][16][17][18][19][20] and PE technology 21 have been applied in hPSCs for either disease modeling or mutation correction.…”

Section: Introductionmentioning

confidence: 99%

Enhancing Precise Genome Editing in Human Pluripotent Stem Cells through Dual Inhibition of DNA Damage Response and Repair Pathways

Park

Kim

Kang

et al. 2023

Preprint

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Precise genome editing is crucial for establishing isogenic human disease models and ex vivo stem cell therapy from the patient-derived human pluripotent stem cells (hPSCs). Unlike Cas9-mediated knock-in, cytosine base editor (CBE) and prime editor (PE) achieve the desirable gene correction without inducing DNA double strand breaks. However, hPSCs possess highly active DNA repair systems and are particularly susceptible to p53-dependent cell death. These unique characteristics impede the efficiency of gene editing in hPSCs. Here, we demonstrate that dual inhibition of p53-mediated cell death and distinct activation of the DNA damage repair system upon DNA damage by CBE or PE additively enhanced editing efficiency in hPSCs. The BE4stem system comprised of dominant negative p53 (p53DD) and three UNG inhibitor (UGI), engineered to specifically diminish base excision repair (BER), improved CBE efficiency in hPSCs. Addition of dominant negative MLH1 to inhibit mismatch repair activity and p53DD in the conventional PE system also significantly enhanced PE efficiency in hPSCs. Thus, combined inhibition of the unique cellular cascades engaged in hPSCs upon gene editing could significantly enhance precise genome editing in these cells.

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“…As a result, gene pencil would be a more suitable option for genetic manipulation in hPSCs compared to gene scissors. Additionally, it is worth mentioning that a stepwise protocol for BEs in hPSCs has been recently updated, for successful base substitution in hPSCs [ 89 ].…”

Section: Pros and Cons Of Bes And Pe In Hpscsmentioning

confidence: 99%

Gene editing with ‘pencil’ rather than ‘scissors’ in human pluripotent stem cells

Park

Lee

et al. 2023

Stem Cell Res Ther

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Owing to the advances in genome editing technologies, research on human pluripotent stem cells (hPSCs) have recently undergone breakthroughs that enable precise alteration of desired nucleotide bases in hPSCs for the creation of isogenic disease models or for autologous ex vivo cell therapy. As pathogenic variants largely consist of point mutations, precise substitution of mutated bases in hPSCs allows researchers study disease mechanisms with “disease-in-a-dish” and provide functionally repaired cells to patients for cell therapy. To this end, in addition to utilizing the conventional homologous directed repair system in the knock-in strategy based on endonuclease activity of Cas9 (i.e., ‘scissors’ like gene editing), diverse toolkits for editing the desirable bases (i.e., ‘pencils’ like gene editing) that avoid the accidental insertion and deletion (indel) mutations as well as large harmful deletions have been developed. In this review, we summarize the recent progress in genome editing methodologies and employment of hPSCs for future translational applications.

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Transition Substitution of Desired Bases in Human Pluripotent Stem Cells with Base Editors: A Step-by-Step Guide

Cited by 4 publications

References 32 publications

Enhancing genome editing in hPSCs through dual inhibition of DNA damage response and repair pathways

Enhancing genome editing in hPSCs through dual inhibition of DNA damage response and repair pathways

Enhancing Precise Genome Editing in Human Pluripotent Stem Cells through Dual Inhibition of DNA Damage Response and Repair Pathways

Gene editing with ‘pencil’ rather than ‘scissors’ in human pluripotent stem cells

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