Base editing‐mediated perturbation of endogenous PKM1/2 splicing facilitates isoform‐specific functional analysis in vitro and in vivo

Abstract

Objectives PKM1 and PKM2, which are generated from the alternative splicing of PKM gene, play important roles in tumourigenesis and embryonic development as rate‐limiting enzymes in glycolytic pathway. However, because of the lack of appropriate techniques, the specific functions of the 2 PKM splicing isoforms have not been clarified endogenously yet. Materials and methods In this study, we used CRISPR‐based base editors to perturbate the endogenous alternative splicing of PKM by introducing mutations into the splicing junction sites in HCT116 cells and zebrafish embryos. Sanger sequencing, agarose gel electrophoresis and targeted deep sequencing assays were utilized for identifying mutation efficiencies and detecting PKM1/2 splicing isoforms. Cell proliferation assays and RNA‐seq analysis were performed to describe the effects of perturbation of PKM1/2 splicing in tumour cell growth and zebrafish embryo development. Results The splicing sites of PKM, a 5’ donor site of GT and a 3’ acceptor site of AG, were efficiently mutated by cytosine base editor (CBE; BE4max) and adenine base editor (ABE; ABEmax‐NG) with guide RNAs (gRNAs) targeting the splicing sites flanking exons 9 and 10 in HCT116 cells and/or zebrafish embryos. The mutations of the 5’ donor sites of GT flanking exons 9 or 10 into GC resulted in specific loss of PKM1 or PKM2 expression as well as the increase in PKM2 or PKM1 respectively. Specific loss of PKM1 promoted cell proliferation of HCT116 cells and upregulated the expression of cell cycle regulators related to DNA replication and cell cycle phase transition. In contrast, specific loss of PKM2 suppressed cell growth of HCT116 cells and resulted in growth retardation of zebrafish. Meanwhile, we found that mutation of PKM1/2 splicing sites also perturbated the expression of non‐canonical PKM isoforms and produced some novel splicing isoforms. Conclusions This work proved that CRISPR‐based base editing strategy can be used to disrupt the endogenous alternative splicing of genes of interest to study the function of specific splicing isoforms in vitro and in vivo. It also reminded us to notice some novel or undesirable splicing isoforms by targeting the splicing junction sites using base editors. In sum, we establish a platform to perturbate endogenous RNA splicing for functional investigation or genetic correction of abnormal splicing events in human diseases.

SMYD3 promotes aerobic glycolysis in diffuse large B-cell lymphoma via H3K4me3-mediated PKM2 transcription

“…The protein expression of PKM1 was very low in DLBCL tissue. Recent study has reported that the expression of PKM1 tends to be cancer-specific and may have tumor-suppressive and tumor-promoting roles based on varying contexts [ 17 ]. Therefore, it is crucial to further investigate the expression and the role of PKM1 in DLBCL cells.…”

“…The reversion of PKM1 to PKM2 is indispensable for shifting glucose metabolism toward aerobic glycolysis [ 16 ]. It has been reported that specific loss of PKM2 suppresses the growth of HCT116 cells [ 17 ]. An increased PKM2/PKM1 ratio has been observed in multiple cancer types and is significantly associated with poor overall survival (OS) [ 18 , 19 ].…”

Two Compact Cas9 Ortholog-Based Cytosine Base Editors Expand the DNA Targeting Scope and Applications In Vitro and In Vivo

“…HEK293T cells were transfected with 1,500 ng ancSgo-BE4 or ancSth1a-BE4, 500 ng gRNA (with a GFP indicator), and 100 ng target reporter using EZtrans reagents in 24-well plates, and three replicates were performed for each group. The cells obtained from cell sorting of GFP/mRuby double-positive cells using flow cytometry were subjected to lysis and PCR amplifications using Phanta Max SuperFidelity DNA polymerase (Vazyme; P505) as we previously described ( Lin et al, 2021 ). The target deep sequencing results for calculating the editing efficiencies.…”

The emerging role of PKM in keratinocyte homeostasis and pathophysiology

“…PKM1 up-regulation in Ex10-mutant cells is corroborated by some degree of induced-whether new or enhanced-PKM1 expression in multiple cell types following PKM2 depletion. For example, PKM1 was upregulated in response to the following PKM2-targeting gene manipulations: CRISPR-guided base editing of the exon 10 3 0 or 5 0 SS in myoblasts, colon cancer cells and zebrafish embryos [26,27]; CRISPR/Cas9-induced double-strand break and mutation of exon 10 in breast cancer cells [28]; and Cre recombinase-mediated deletion of exon 10 in mouse embryonic fibroblasts [29]. However, when utilizing the same gene-editing technique, the extent of PKM1 induction could be tissue-or cell-specific.…”