Multiplexed base editing through Cas12a variant-mediated cytosine and adenine base editors

被引:24
作者
Chen, Fangbing [1 ]
Lian, Meng [1 ,2 ,3 ]
Ma, Bingxiu [4 ]
Gou, Shixue [1 ,5 ]
Luo, Xian [6 ]
Yang, Kaiming [1 ,7 ]
Shi, Hui [1 ,5 ]
Xie, Jingke [1 ,5 ,6 ]
Ge, Weika [1 ,5 ,6 ]
Ouyang, Zhen [1 ,3 ,5 ,6 ,8 ]
Lai, Chengdan [1 ,3 ,5 ,6 ,8 ]
Li, Nan [1 ,5 ,6 ]
Zhang, Quanjun [1 ,3 ,5 ,8 ]
Jin, Qin [1 ,3 ,5 ,8 ]
Liang, Yanhui [1 ,5 ]
Chen, Tao [6 ]
Wang, Jiaowei [1 ,9 ]
Zhao, Xiaozhu [1 ,9 ]
Li, Lei [1 ,9 ]
Yu, Manya [1 ,7 ]
Ye, Yinghua [1 ,3 ,5 ,8 ]
Wang, Kepin [1 ,3 ,5 ,6 ,8 ]
Wu, Han [1 ,3 ,5 ,8 ]
Lai, Liangxue [1 ,3 ,5 ,6 ,7 ,8 ]
机构
[1] Chinese Acad Sci, Guangzhou Inst Biomed & Hlth, Guangdong Prov Key Lab Stem Cell & Regenerat Med, CAS Key Lab Regenerat Biol, Guangzhou 510530, Peoples R China
[2] Chinese Acad Med Sci & Peking Union Med Coll, Inst Lab Anim Sci, Beijing 100730, Peoples R China
[3] Chinese Acad Med Sci, Res Unit Generat Large Anim Dis Models, 2019RU015, Guangzhou 510530, Peoples R China
[4] Guangzhou Med Univ, Affiliated Hosp 3, Key Lab Reprod & Genet Guangdong Higher Educ Inst, Dept Gynecol & Obstet,Key Lab Major Obstetr Dis G, Guangzhou 510150, Peoples R China
[5] Hainan Prov Res Ctr Lab Anim, Sanya Inst Swine Resource, Sanya 572000, Peoples R China
[6] Wuyi Univ, Guangdong Prov Key Lab Large Anim Models Biomed, Jiangmen 529020, Peoples R China
[7] Guangzhou Med Univ, Chinese Acad Sci, Guangzhou Inst Biomed & Hlth, Joint Sch Life Sci, Guangzhou 511436, Peoples R China
[8] Guangzhou Regenerat Med & Hlth Guangdong Lab, Bioland Lab, Guangzhou 510005, Peoples R China
[9] Univ Chinese Acad Sci, Beijing 100049, Peoples R China
基金
中国国家自然科学基金;
关键词
GENOMIC DNA; OFF-TARGET; CPF1;
D O I
10.1038/s42003-022-04152-8
中图分类号
Q [生物科学];
学科分类号
07 ; 0710 ; 09 ;
摘要
Cas12a-mediated cytosine (CBE) and adenine base editor (ABE) systems with elevated efficiencies and expanded target scopes are developed, by combining highly active deaminases with Cas12a variants, enCas12a, RR and RVR, recognizing non-TTTV PAMs. Cas12a can process multiple sgRNAs from a single transcript of CRISPR array, conferring advantages in multiplexed base editing when incorporated into base editor systems, which is extremely helpful given that phenotypes commonly involve multiple genes or single-nucleotide variants. However, multiplexed base editing through Cas12a-derived base editors has been barely reported, mainly due to the compromised efficiencies and restricted protospacer-adjacent motif (PAM) of TTTV for wild-type Cas12a. Here, we develop Cas12a-mediated cytosine base editor (CBE) and adenine base editor (ABE) systems with elevated efficiencies and expanded targeting scope, by combining highly active deaminases with Lachnospiraceae bacterium Cas12a (LbCas12a) variants. We confirm that these CBEs and ABEs can perform efficient C-to-T and A-to-G conversions, respectively, on targets with PAMs of NTTN, TYCN, and TRTN. Notably, multiplexed base editing can be conducted using the developed CBEs and ABEs in somatic cells and embryos. These Cas12a variant-mediated base editors will serve as versatile tools for multiplexed point mutation, which is notably important in genetic improvement, disease modeling, and gene therapy.
引用
收藏
页数:12
相关论文
共 51 条
[1]   Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases [J].
Bae, Sangsu ;
Park, Jeongbin ;
Kim, Jin-Soo .
BIOINFORMATICS, 2014, 30 (10) :1473-1475
[2]   Multiplexed orthogonal genome editing and transcriptional activation by Cas12a [J].
Breinig, Marco ;
Schweitzer, Anabel Y. ;
Herianto, Anna M. ;
Revia, Steffie ;
Schaefer, Lisa ;
Wendler, Lena ;
Galvez, Ana Cobos ;
Tschaharganeh, Darjus F. .
NATURE METHODS, 2019, 16 (01) :51-+
[3]   Induction of Chromosomal Translocations with CRISPR-Cas9 and Other Nucleases: Understanding the Repair Mechanisms That Give Rise to Translocations [J].
Brunet, Erika ;
Jasin, Maria .
CHROMOSOME TRANSLOCATION, 2018, 1044 :15-25
[4]   Multiplexed genome engineering by Cas12a and CRISPR arrays encoded on single transcripts [J].
Campa, Carlo C. ;
Weisbach, Niels R. ;
Santinha, Antonio J. ;
Incarnato, Danny ;
Platt, Randall J. .
NATURE METHODS, 2019, 16 (09) :887-+
[5]   Robustly improved base editing efficiency of Cpf1 base editor using optimized cytidine deaminases [J].
Chen, Siyu ;
Jia, Yingqi ;
Liu, Zhiquan ;
Shan, Huanhuan ;
Chen, Mao ;
Yu, Hao ;
Lai, Liangxue ;
Li, Zhanjun .
CELL DISCOVERY, 2020, 6 (01)
[6]   CRISPResso2 provides accurate and rapid genome editing sequence analysis [J].
Clement, Kendell ;
Rees, Holly ;
Canver, Matthew C. ;
Gehrke, Jason M. ;
Farouni, Rick ;
Hsu, Jonathan Y. ;
Cole, Mitchel A. ;
Liu, David R. ;
Joung, J. Keith ;
Bauer, Daniel E. ;
Pinello, Luca .
NATURE BIOTECHNOLOGY, 2019, 37 (03) :224-226
[7]   Evaluation and minimization of Cas9-independent off-target DNA editing by cytosine base editors [J].
Doman, Jordan L. ;
Raguram, Aditya ;
Newby, Gregory A. ;
Liu, David R. .
NATURE BIOTECHNOLOGY, 2020, 38 (05) :620-+
[8]   The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA [J].
Fonfara, Ines ;
Richter, Hagen ;
Bratovic, Majda ;
Le Rhun, Anais ;
Charpentier, Emmanuelle .
NATURE, 2016, 532 (7600) :517-+
[9]   Engineered Cpf1 variants with altered PAM specificities [J].
Gao, Linyi ;
Cox, David B. T. ;
Yan, Winston X. ;
Manteiga, John C. ;
Schneider, Martin W. ;
Yamano, Takashi ;
Nishimasu, Hiroshi ;
Nureki, Osamu ;
Crosetto, Nicola ;
Zhang, Feng .
NATURE BIOTECHNOLOGY, 2017, 35 (08) :789-792
[10]   Directed evolution of adenine base editors with increased activity and therapeutic application [J].
Gaudelli, Nicole M. ;
Lam, Dieter K. ;
Rees, Holly A. ;
Sola-Esteves, Noris M. ;
Barrera, Luis A. ;
Born, David A. ;
Edwards, Aaron ;
Gehrke, Jason M. ;
Lee, Seung-Joo ;
Liquori, Alexander J. ;
Murray, Ryan ;
Packer, Michael S. ;
Rinaldi, Conrad ;
Slaymaker, Ian M. ;
Yen, Jonathan ;
Young, Lauren E. ;
Ciaramella, Giuseppe .
NATURE BIOTECHNOLOGY, 2020, 38 (07) :892-U99