Efficient base editing for multiple genes and loci in pigs using base editors

Abstract

Cytosine base editors (CBEs) enable programmable C-toT conversion without DNA doublestranded breaks and homology-directed repair in a variety of organisms, which exhibit great potential for agricultural and biomedical applications. However, all reported cases only involved C-toT substitution at a single targeted genomic site. Whether C-toT substitution is effective in multiple sites/loci has not been verified in large animals. Here, by using pigs, an important animal for agriculture and biomedicine, as the subjective animal, we showed that CBEs could efficiently induce C-toT conversions at multiple sites/loci with the combination of three genes, including DMD, TYR, and LMNA, or RAG1, RAG2, and IL2RG, simultaneously, at the embryonic and cellular levels. CBEs also could disrupt genes (pol gene of porcine endogenous retrovirus) with dozens of copies by introducing multiple premature stop codons. With the CBEs, pigs carrying single gene or multiple gene point mutations were generated through embryo injection or nuclear transfer approach.

Chemical modifications of adenine base editor mRNA and guide RNA expand its application scope

“…By applying various chemical modifications to ABE mRNA and guide RNA, we successfully engineered an RNA-encoded base editing system. Although previous reports show that unmodified cytidine base editor mRNA and guide RNA could effectively edit embryos and oocytes 14,15 , our data show that unmodified ABE mRNA does not effectively express in HEK293T cells and unmodified guide RNA cannot mediate efficient editing in somatic cell culture. This might be because unmodified mRNA and guide RNA are not stable and will quickly undergo degradation after being delivered to cell culture.…”

“…Microinjection of cytidine base editor mRNA and in vitro transcribed guide RNA can introduce effective base editing in mouse embryo or pig oocyte 14,15 , suggesting the potential of using RNA-encoded base editors to mediate editing. However, the editing efficiency mediated by RNA-encoded ABE system has not been studied in somatic cells, and its application potential and delivery method have not been addressed.…”

A Tale of Two Moieties: Rapidly Evolving CRISPR/Cas-Based Genome Editing

“…For higher C-to-T editing efficiency, rA1 was replaced by other types of APOBEC deaminases, which also expands the editing scope [50,62]. For instance, conjugating human APOBEC3A (hA3A) in CBEs can efficiently edit cytosines in highly methylated regions and in the GpC dinucleotide content [63][64][65][66][67]. Furthermore, fusing engineered and/or in vitro evolved APOBEC effectors (e.g., rA1-YE1, rA1-YEE, hA3A-Y130F, hA3A-Y132D, eA3A and evoA1) in CBEs can narrow the base editing window (a context region within gRNA target site in which all cytosines can be potentially converted to thymines) to reduce unintended bystander mutations (unintended C-to-T changes within editing windows) and to diversify editing scopes for C-to-T changes [63,[68][69][70].…”

Base editing in pigs for precision breeding

“…Furthermore, a study reported that CBEs efficiently induced C-to-T conversions of triple genes simultaneously, including RAG1, RAG2, and IL2RG, or DMD, TYR, and LMNA. These findings will help to accelerate the generation of animal models with multiplex point mutations and studies in gene therapies of genetic diseases [101] . The pig model with RAG1, RAG2, and IL2RG mutations lacked B cells, T cells, and NK cells, providing a great prospect for xenotransplantation.…”