Highly elevated base excision repair pathway in primordial germ cells causes low base editing activity in chickens

Abstract

Base editing technology enables the generation of precisely genome-modified animal models. In this study, we applied base editing to chicken, an important livestock animal in the fields of agriculture, nutrition, and research through primordial germ cell (PGC)-mediated germline transmission. Using this approach, we successfully produced two genome-modified chicken lines harboring mutations in the genes encoding ovotransferrin (TF) and myostatin (MSTN); however, only 55.5% and 35.7% of genome-modified chickens had the desired base substitutions in TF and MSTN, respectively. To explain the low base-editing activity, we performed molecular analysis to compare DNA repair pathways between PGCs and the chicken fibroblast cell line DF-1. The results revealed that base excision repair (BER)-related genes were significantly elevated in PGCs relative to DF-1 cells. Subsequent functional studies confirmed that the editing activity could be regulated by modulating the expression of uracil N-glycosylase (UNG), an upstream gene of the BER pathway. Collectively, our findings indicate that the distinct DNA repair property of chicken PGCs causes low editing activity during genome modification, however, modulation of BER functions could promote the production of genome-modified organisms with the desired genotypes. K E Y W O R D S base editing, base excision repair, chicken, editing activity, primordial germ cell, uracil N-glycosylase 2 | LEE Et aL.

Chicken blastoderms and primordial germ cells possess a higher expression of DNA repair genes and lower expression of apoptosis genes to preserve their genome stability

“…Likewise, there is only one report on the expression of DNA repair (BER) pathway genes in the chicken (primordial) germ cells. In that study, several BER pathway genes were significantly upregulated in PGCs than CEF cell line (DF-1) by RT-qPCR analysis, and subsequent functional studies confirmed that the base editing activity in PGCs could be regulated by modulating the expression of UNG , an upstream gene of the BER pathway 35 . The results that the higher expression of apoptosis genes in the normal CEFs could be a potential reason for hampering the long-term culture of primary CEFs 12 .…”

Prime editing in chicken fibroblasts and primordial germ cells

“…The proposed method would provide precise genome editing in PGCs and enable tests for gene function in PGC development in both in vitro and in vivo settings. Furthermore, because the PGC-mediated germline transmission system is widely used to generate transgenic chickens (van de Lavoir et al, 2006;Macdonald et al, 2010;Macdonald et al, 2012;Oishi et al, 2016;Kim et al, 2020;Lee et al, 2020;Park et al, 2020), our method could underlie potential applications for the efficient genomic modification of chickens. methodology; writing-review & editing.…”

“…TG chickens have great potential not only as a useful model for developmental biology but also as a bioreactor in the agricultural and pharmaceutical fields. Currently, CRISPR-based technologies, including base editing, have been used for genome modification of chicken PGCs (Dimitrov et al ., 2016; Idoko-Akoh et al ., 2018; Kim et al ., 2020; Lee et al ., 2020; Park et al ., 2020). However, PE has not yet been applied to chicken PGCs.…”

Efficient gene transfer into zebra finch germline-competent stem cells using an adenoviral vector system

“…The primary culture of ZEFs was prepared from the muscles of 6-day-old zebra finch embryos 43 . Several embryos (n = 3–5) were extracted from fertilized eggs, their head, limbs, and organs were removed, and the remaining contents were minced.…”