Programmable base editing efficiency study of CRISPR/Cas9-guided DNA base editors in pig genome

doi:10.14088/j.cnki.issn0439-8114.2020.18.029

Abstract

Abstract: The CRISPR/Cas9-based BE3（Cytosine base editor,CBE） and ABE7.10（Adenine base editor,ABE） were used to analyze and study the editing efficiency of target gene loci in pig genome. Four porcine genomic target gene loci gRNA expression vectors were designed, synthesized and constructed, which were co-transfected into PK15 cells with CBE or ABE, respectively. The PK15 cells were further cultured for 48 h, and single-base replacement efficiency and indels incidence were determined by second-generation sequencing technology. The results showed that the active editing Windows of the single base editing system BE3 and ABE7.10 were 5 nucleotides and 4 nucleotides, respectively; Both BE3 and ABE7.10 were mainly contributed to single base conversion but not indel; The two sets of single-base editing systems showed certain preference for base replacement in pig genome. In the CMAH, MC1R（1-2） and MC1R（2-1） gene editing Windows, the efficiency of C→T was 2.2%, 0.4% and 1.3%, respectively; The efficiency of A→G in pig GGTA and MSTN-2 was 1.4% and 1.4%, respectively. It is shown that both BE3 and ABE7.10 can perform effective single base substitutions for genomic target sequences in pig cells.

Key words: pig, single base editing, CRISPR/Cas9, BE3, ABE7.10

CLC Number:

Q812

ZHANG Ting-ting, CHEN Tao, LI Yan-li, YANG Man-man, WEI Qiang, WANG Ran, LI Lin, LI Yong. Programmable base editing efficiency study of CRISPR/Cas9-guided DNA base editors in pig genome[J]. HUBEI AGRICULTURAL SCIENCES, 2020, 59(18): 143-149.

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