scholarly journals Adenine base-editing-mediated exon skipping induces gene knockout in cultured pig cells

2022 ◽  
Author(s):  
Xiang-xing Zhu ◽  
Jia-sheng Pan ◽  
Tao Lin ◽  
Ye-cheng Yang ◽  
Qiu-yan Huang ◽  
...  
Keyword(s):  
Gene Knockout ◽  
Exon Skipping ◽  
Base Editing ◽  
Adenine Base

scholarly journals Adenine Base-Editing-Mediated Exon Skipping Induces Gene Knockout in Cultured Pig Cells

2021 ◽  
Author(s):  
Xiang-xing Zhu ◽  
Jia-sheng Pan ◽  
Tao Lin ◽  
Ye-cheng Yang ◽  
Qiu-yan Huang ◽  
...  
Keyword(s):  
Genetic Modification ◽  
Gene Knockout ◽  
Exon Skipping ◽  
Splice Acceptor Site ◽  
Splice Donor Site ◽  
Acceptor Site ◽  
Splice Acceptor ◽  
Single Base ◽  
Base Editing ◽  
Adenine Base

Abstract Gene-knockout pigs have important applications in agriculture and medicine. Compared with CRISPR/Cas9, Adenine base editor (ABE) convert single A·T pairs to G·C pairs in the genome without generating DNA double-strand breaks, and this method has higher accuracy and biosafety in pig genetic modification. However, the application of ABE in pig gene knockout is limited by protospacer-adjacent motif (PAM) sequences and the base-editing window. Alternative mRNA splicing is an important mechanism underlying the formation of proteins with diverse functions in eukaryotes. Spliceosome recognizes the conservative sequences of splice donors and acceptors in a precursor mRNA. Mutations in these conservative sequences induce exon skipping, leading to proteins with novel functions or to gene inactivation due to frameshift mutations. In this study, adenine base-editing-mediated exon skipping was used to expand the application of ABE in the generation of gene knockout pigs. We first constructed a modified “all-in-one” ABE vector suitable for porcine somatic cell transfection that contained an ABE for single-base editing and an sgRNA expression cassette. The “all-in-one” ABE vector induced efficient sgRNA-dependent A-to-G conversions in porcine cells during single base-editing of multiple endogenous gene loci. Subsequently, an ABE system was designed for single adenine editing of the conservative splice acceptor site (AG sequence at the 3’ end of the intron 5) and splice donor site (GT sequence at the 5’ end of the intron 6) in the porcine gene GHR; this method achieved highly efficient A-to-G conversion at the cellular level. Then, porcine single-cell colonies carrying a biallelic A-to-G conversion in the splice acceptor site in the intron 5 of GHR were generated. RT-PCR indicated exon 6 skipped at the mRNA level. Western blotting revealed GHR protein loss, and gene sequencing showed no sgRNA-dependent off-target effects. These results demonstrate accurate adenine base-editing-mediated exon skipping and gene knockout in porcine cells. This is the first proof-of-concept study of adenine base-editing-mediated exon skipping for gene regulation in pigs, and this work provides a new strategy for accurate and safe genetic modification of pigs for agricultural and medical applications.

scholarly journals Adenine Base-Editing-Mediated Exon Skipping Induces Gene Knockout in Cultured Pig Cells

2021 ◽  
Author(s):  
XIANGXING ZHU ◽  
Jia-sheng Pan ◽  
Tao Lin ◽  
Ye-cheng Yang ◽  
Qiu-yan Huang ◽  
...  
Keyword(s):  
Genetic Modification ◽  
Gene Knockout ◽  
Exon Skipping ◽  
Splice Acceptor Site ◽  
Splice Donor Site ◽  
Acceptor Site ◽  
Splice Acceptor ◽  
Single Base ◽  
Base Editing ◽  
Adenine Base

Abstract Gene-knockout pigs have important applications in agriculture and medicine. Compared with CRISPR/Cas9, Adenine base editor (ABE) convert single A·T pairs to G·C pairs in the genome without generating DNA double-strand breaks, and this method has higher accuracy and biosafety in pig genetic modification. However, the application of ABE in pig gene knockout is limited by protospacer-adjacent motif (PAM) sequences and the base-editing window. Alternative mRNA splicing is an important mechanism underlying the formation of proteins with diverse functions in eukaryotes. Spliceosome recognizes the conservative sequences of splice donors and acceptors in a precursor mRNA. Mutations in these conservative sequences induce exon skipping, leading to proteins with novel functions or to gene inactivation due to frameshift mutations. In this study, adenine base-editing-mediated exon skipping was used to expand the application of ABE in the generation of gene knockout pigs. We first constructed a modified “all-in-one” ABE vector suitable for porcine somatic cell transfection that contained an ABE for single-base editing and an sgRNA expression cassette. The “all-in-one” ABE vector induced efficient sgRNA-dependent A-to-G conversions in porcine cells during single base-editing of multiple endogenous gene loci. Subsequently, an ABE system was designed for single adenine editing of the conservative splice acceptor site (AG sequence at the 3’ end of the intron 5) and splice donor site (GT sequence at the 5’ end of the intron 6) in the porcine gene GHR; this method achieved highly efficient A-to-G conversion at the cellular level. Then, porcine single-cell colonies carrying a biallelic A-to-G conversion in the splice acceptor site in the intron 5 of GHR were generated. RT-PCR indicated exon 6 skipped at the mRNA level. Western blotting revealed GHR protein loss, and gene sequencing showed no sgRNA-dependent off-target effects. These results demonstrate accurate adenine base-editing-mediated exon skipping and gene knockout in porcine cells. This is the first proof-of-concept study of adenine base-editing-mediated exon skipping for gene regulation in pigs, and this work provides a new strategy for accurate and safe genetic modification of pigs for agricultural and medical applications.

scholarly journals Effective and precise adenine base editing in mouse zygotes

2018 ◽  
Vol 9 (9) ◽  
pp. 808-813 ◽  
Author(s):  
Puping Liang ◽  
Hongwei Sun ◽  
Xiya Zhang ◽  
Xiaowei Xie ◽  
Jinran Zhang ◽  
...  
Keyword(s):  
Base Editing ◽  
Mouse Zygotes ◽  
Adenine Base

Adenine Base Editor Ribonucleoproteins Delivered by Lentivirus-Like Particles Show High On-Target Base Editing and Undetectable RNA Off-Target Activities

2021 ◽  
Vol 4 (1) ◽  
pp. 69-81 ◽  
Author(s):  
Pin Lyu ◽  
Zuyan Lu ◽  
Sung-Ik Cho ◽  
Manish Yadav ◽  
Kyung Whan Yoo ◽  
...  
Keyword(s):  
Base Editing ◽  
Adenine Base

scholarly journals Rescue of STAT3 Function in Hyper-IgE Syndrome Using Adenine Base Editing

2021 ◽  
Vol 4 (2) ◽  
pp. 178-190
Author(s):  
Andreas C. Eberherr ◽  
Andre Maaske ◽  
Christine Wolf ◽  
Florian Giesert ◽  
Riccardo Berutti ◽  
...  
Keyword(s):  
Base Editing ◽  
Hyper Ige Syndrome ◽  
Adenine Base

scholarly journals Cytosine base editor 4 but not adenine base editor generates off-target mutations in mouse embryos

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Hye Kyung Lee ◽  
Harold E. Smith ◽  
Chengyu Liu ◽  
Michaela Willi ◽  
Lothar Hennighausen
Keyword(s):  
Point Mutations ◽  
Mouse Embryos ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Base Editing ◽  
Genome Wide ◽  
Wide Range ◽  
Family Based ◽  
Correct Point ◽  
Adenine Base

AbstractDeaminase base editing has emerged as a tool to install or correct point mutations in the genomes of living cells in a wide range of organisms. However, the genome-wide off-target effects introduced by base editors in the mammalian genome have been examined in only one study. Here, we have investigated the fidelity of cytosine base editor 4 (BE4) and adenine base editors (ABE) in mouse embryos using unbiased whole-genome sequencing of a family-based trio cohort. The same sgRNA was used for BE4 and ABE. We demonstrate that BE4-edited mice carry an excess of single-nucleotide variants and deletions compared to ABE-edited mice and controls. Therefore, an optimization of cytosine base editors is required to improve its fidelity. While the remarkable fidelity of ABE has implications for a wide range of applications, the occurrence of rare aberrant C-to-T conversions at specific target sites needs to be addressed.

scholarly journals Author Correction: Precision genome engineering through adenine base editing in plants

Nature Plants ◽  
2018 ◽  
Vol 4 (9) ◽  
pp. 730-730
Author(s):  
Beum-Chang Kang ◽  
Jae-Young Yun ◽  
Sang-Tae Kim ◽  
YouJin Shin ◽  
Jahee Ryu ◽  
...  
Keyword(s):  
Genome Engineering ◽  
Base Editing ◽  
Adenine Base

scholarly journals Docking sites inside Cas9 for adenine base editing diversification and RNA off-target elimination

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Shuo Li ◽  
Bo Yuan ◽  
Jixin Cao ◽  
Jingqi Chen ◽  
Jinlong Chen ◽  
...  
Keyword(s):  
Streptococcus Pyogenes ◽  
Base Editing ◽  
The Future ◽  
Docking Sites ◽  
Domain Insertion ◽  
Adenine Base

AbstractBase editing tools with diversified editing scopes and minimized RNA off-target activities are required for broad applications. Nevertheless, current Streptococcus pyogenes Cas9 (SpCas9)-based adenine base editors (ABEs) with minimized RNA off-target activities display constrained editing scopes with efficient editing activities at positions 4-8. Here, functional ABE variants with diversified editing scopes and reduced RNA off-target activities are identified using domain insertion profiling inside SpCas9 and with different combinations of TadA variants. Engineered ABE variants in this study display narrowed, expanded or shifted editing scopes with efficient editing activities across protospacer positions 2-16. And when combined with deaminase engineering, the RNA off-target activities of engineered ABE variants are further minimized. Thus, domain insertion profiling provides a framework to improve and expand ABE toolkits, and its combination with other strategies for ABE engineering deserves comprehensive explorations in the future.

scholarly journals Adenine Base Editing in vivo with a Single Adeno-Associated Virus Vector

2021 ◽  
Author(s):  
Han Zhang ◽  
Nathan Bamidele ◽  
Pengpeng Liu ◽  
Ogooluwa Ojelabi ◽  
Xin D. Gao ◽  
...  
Keyword(s):  
Genetic Diseases ◽  
Cell Types ◽  
Vector System ◽  
Adeno Associated Virus ◽  
Base Editing ◽  
Guide Rna ◽  
Gene Therapies ◽  
Compact Size ◽  
Adenine Base

Base editors (BEs) have opened new avenues for the treatment of genetic diseases. However, advances in delivery approaches are needed to enable disease targeting of a broad range of tissues and cell types. Adeno-associated virus (AAV) vectors remain one of the most promising delivery vehicles for gene therapies. Currently, most BE/guide combinations and their promoters exceed the packaging limit (~5 kb) of AAVs. Dual-AAV delivery strategies often require high viral doses that impose safety concerns. In this study, we engineered an adenine base editor using a compact Cas9 from Neisseria meningitidis (Nme2Cas9). Compared to the well-characterized Streptococcus pyogenes Cas9-containing ABEs, Nme2-ABE possesses a distinct PAM (N4CC) and editing window, exhibits fewer off-target effects, and can efficiently install therapeutically relevant mutations in both human and mouse genomes. Importantly, we showed that in vivo delivery of Nme2-ABE and its guide RNA by a single-AAV vector can revert the disease mutation and phenotype in an adult mouse model of tyrosinemia. We anticipate that Nme2-ABE, by virtue of its compact size and broad targeting range, will enable a range of therapeutic applications with improved safety and efficacy due in part to packaging in a single-vector system.

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