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

Abstract

The recent advances in human pluripotent stem cells (hPSCs) enable to precisely edit the desired bases in hPSCs to be used for the establishment of isogenic disease models and autologous ex vivo cell therapy. The knock-in approach based on the homologous directed repair with Cas9 endonuclease, causing DNA double-strand breaks (DSBs), produces not only insertion and deletion (indel) mutations but also deleterious large deletions. On the contrary, due to the lack of Cas9 endonuclease activity, base editors (BEs) such as adenine base editor (ABE) and cytosine base editor (CBE) allow precise base substitution by conjugated deaminase activity, free from DSB formation. Despite the limitation of BEs in transition substitution, precise base editing by BEs with no massive off-targets is suggested to be a prospective alternative in hPSCs for clinical applications. Considering the unique cellular characteristics of hPSCs, a few points should be considered. Herein, we describe an updated and optimized protocol for base editing in hPSCs. We also describe an improved methodology for CBE-based C to T substitutions, which are generally lower than A to G substitutions in hPSCs.

Keywords: Base editors; Cas9; Genome editing; Human pluripotent stem cells; Transition substitution.

Fig. 1

Composition and mechanisms of ABE8e and AncBE4max. Graphical scheme for composition and mechanisms of (A) ABE8e and (B) AncBE4max, Red box indicates editing window, bases, colored in blue indicates spacer sequence, PAM sequence is colored in red, and the target base for each base editor is colored in yellow. Number indicates the position in the spacer sequence. A for Adenine, I for Inosine, G for Guanine, C for Cytosine, U for Uracil land T for Thymine. Created with BioRender.com.

Fig. 2

Role of DNA replication, mismatch repair, base excision repair and UNG in C to T conversion. (i) Nickase activity of nCas9 in CBE induces nick on the editing strand and deaminase activity in CBE produces G:U mismatch. G:U mismatch is converted to A:T via DNA replication followed by mismatch repair. (ii) Alternatively, G:U mismatch, recognized by base excision repair (BER), is removed by UNG to produce AP site, forming G:C. (iii) UNG activity to impair the G-to-A substitution is the co-expression of UNG inhibitor (UGI). Created with BioRender.com.

Fig. 3

PAM requirement for base editing. (A) Graphical scheme for editing window of BEs, Red box indicates editing window. ‘x’ and ‘y’ indicates start and end position of editing window respectively. (B) Target base is colored in red and PAM sequence is colored in brown. Graphical scheme of PAM requirement for, (C) ABE8e, (D) YE1-BE4max, (E) NG-ABE8e, and (F) YE1-BE4max-NG. Target base is colored in red and PAM sequence is colored in brown. Created with BioRender.com.

Fig. 4

Bystander base editing of AncBE4max. (A) Graphical scheme for the bystander base in the editing window (colored in red) and possible outcomes. (B) Sequences of GNE encoding 329 isoleucine (329I) in WT hESCs (i), GNE mutants hESCs after ABE application (ii), isolated hESCs with I329I silence mutation due to bystander editing (blue arrow) and GNE mutant hESCs with I329T mutation from target base edit (red arrow) (C) Graphical scheme for GNE mutant hPSCs, WT (blue), I329I mutant (red), and I329T mutant hESCs (green). Created with BioRender.com.