MutSα and MutSβ as size-dependent cellular determinants for prime editing in human embryonic stem cells

Ju-Chan Park; Yun-Jeong Kim; Jun Hee Han; Dayeon Kim; Mihn Jeong Park; Jumee Kim; Hyeon-Ki Jang; Sangsu Bae; Hyuk-Jin Cha

doi:10.1016/j.omtn.2023.05.015

MutSα and MutSβ as size-dependent cellular determinants for prime editing in human embryonic stem cells

Mol Ther Nucleic Acids. 2023 May 18:32:914-922. doi: 10.1016/j.omtn.2023.05.015. eCollection 2023 Jun 13.

Authors

Ju-Chan Park¹, Yun-Jeong Kim¹, Jun Hee Han², Dayeon Kim¹, Mihn Jeong Park¹, Jumee Kim¹, Hyeon-Ki Jang³, Sangsu Bae⁴, Hyuk-Jin Cha^{1

5}

Affiliations

¹ College of Pharmacy, Seoul National University, Seoul, Republic of Korea.
² Department of Chemistry, Hanyang University, Seoul, Republic of Korea.
³ Division of Chemical Engineering and Bioengineering, College of Art Culture and Engineering, Kangwon National University, Chuncheon, South Korea.
⁴ College of Medicine, Seoul National University, Seoul, Republic of Korea.
⁵ Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, Republic of Korea.

Abstract

Precise genome editing in human pluripotent stem cells (hPSCs) has potential applications in isogenic disease modeling and ex vivo stem cell therapy, necessitating diverse genome editing tools. However, unlike differentiated somatic cells, hPSCs have unique cellular properties that maintain genome integrity, which largely determine the overall efficiency of an editing tool. Considering the high demand for prime editors (PEs), it is imperative to characterize the key molecular determinants of PE outcomes in hPSCs. Through homozygous knockout (KO) of MMR pathway key proteins MSH2, MSH3, and MSH6, we reveal that MutSα and MutSβ determine PE efficiency in an editing size-dependent manner. Notably, MSH2 perturbation disrupted both MutSα and MutSβ complexes, dramatically escalating PE efficiency from base mispair to 10 bases, up to 50 folds. Similarly, impaired MutSα by MSH6 KO improved editing efficiency from single to three base pairs, while defective MutSβ by MSH3 KO heightened efficiency from three to 10 base pairs. Thus, the size-dependent effect of MutSα and MutSβ on prime editing implies that MMR is a vital PE efficiency determinant in hPSCs and highlights the distinct roles of MutSα and MutSβ in its outcome.

Keywords: DNA damage repair; MMR; MT: RNA/DNA editing;; MutSα; MutSβ; genome editing; human embryonic stem cells; human pluripotent stem cells; prime editor.