Miniature type V-F CRISPR-Cas nucleases enable targeted DNA modification in cells

被引:76
作者
Bigelyte, Greta [1 ]
Young, Joshua K. [2 ]
Karvelis, Tautvydas [1 ]
Budre, Karolina [1 ]
Zedaveinyte, Rimante [1 ]
Djukanovic, Vesna [2 ]
Van Ginkel, Elizabeth [2 ]
Paulraj, Sushmitha [2 ]
Gasior, Stephen [2 ]
Jones, Spencer [2 ]
Feigenbutz, Lanie [2 ]
St Clair, Grace [2 ]
Barone, Pierluigi [2 ]
Bohn, Jennifer [2 ]
Acharya, Ananta [2 ]
Zastrow-Hayes, Gina [2 ]
Henkel-Heinecke, Selgar [3 ]
Silanskas, Arunas [1 ]
Seidel, Ralf [3 ]
Siksnys, Virginijus [1 ]
机构
[1] Vilnius Univ, Life Sci Ctr, Inst Biotechnol, Vilnius, Lithuania
[2] Corteva Agrisci, Mol Engn, Johnston, IA 50131 USA
[3] Univ Leipzig, Inst Expt Phys, Leipzig, Germany
基金
欧盟地平线“2020”;
关键词
NUCLEIC-ACID DETECTION; THERMOTOLERANCE; ENDONUCLEASE; MUTAGENESIS; CLEAVAGE; SYSTEM;
D O I
10.1038/s41467-021-26469-4
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
Class 2 CRISPR systems are exceptionally diverse, nevertheless, all share a single effector protein that contains a conserved RuvC-like nuclease domain. Interestingly, the size of these CRISPR-associated (Cas) nucleases ranges from >1000 amino acids (aa) for Cas9/Cas12a to as small as 400-600 aa for Cas12f. For in vivo genome editing applications, compact RNA-guided nucleases are desirable and would streamline cellular delivery approaches. Although miniature Cas12f effectors have been shown to cleave double-stranded DNA, targeted DNA modification in eukaryotic cells has yet to be demonstrated. Here, we biochemically characterize two miniature type V-F Cas nucleases, SpCas12f1 (497 aa) and AsCas12f1 (422 aa), and show that SpCas12f1 functions in both plant and human cells to produce targeted modifications with outcomes in plants being enhanced with short heat pulses. Our findings pave the way for the development of miniature Cas12f1-based genome editing tools. Miniature Cas12f editing systems are well suited for in vivo editing applications. Here the authors characterize the intrinsic activity of SpCas12f1 in plant and animal cells.
引用
收藏
页数:8
相关论文
共 46 条
[1]  
Acharya A., **DATA OBJECT**, DOI 10.5281/zenodo.5237191
[2]   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
[3]   Efficient Gene Targeting in Maize Using Inducible CRISPR-Cas9 and Marker-free Donor Template [J].
Barone, Pierluigi ;
Wu, Emily ;
Lenderts, Brian ;
Anand, Ajith ;
Gordon-Kamm, William ;
Svitashev, Sergei ;
Kumar, Sandeep .
MOLECULAR PLANT, 2020, 13 (08) :1219-1227
[4]   CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity [J].
Chen, Janice S. ;
Ma, Enbo ;
Harrington, Lucas B. ;
Da Costa, Maria ;
Tian, Xinran ;
Palefsky, Joel M. ;
Doudna, Jennifer A. .
SCIENCE, 2018, 360 (6387) :436-+
[5]   Multiplex Genome Engineering Using CRISPR/Cas Systems [J].
Cong, Le ;
Ran, F. Ann ;
Cox, David ;
Lin, Shuailiang ;
Barretto, Robert ;
Habib, Naomi ;
Hsu, Patrick D. ;
Wu, Xuebing ;
Jiang, Wenyan ;
Marraffini, Luciano A. ;
Zhang, Feng .
SCIENCE, 2013, 339 (6121) :819-823
[6]   Male-sterile maize plants produced by targeted mutagenesis of the cytochrome P450-like gene (MS26) using a re-designed I-CreI homing endonuclease [J].
Djukanovic, Vesna ;
Smith, Jeff ;
Lowe, Keith ;
Yang, Meizhu ;
Gao, Huirong ;
Jones, Spencer ;
Nicholson, Michael G. ;
West, Ande ;
Lape, Janel ;
Bidney, Dennis ;
Falco, Saverio Carl ;
Jantz, Derek ;
Lyznik, Leszek Alexander .
PLANT JOURNAL, 2013, 76 (05) :888-899
[7]   Post-Domestication Selection in the Maize Starch Pathway [J].
Fan, Longjiang ;
Bao, Jiandong ;
Wang, Yu ;
Yao, Jianqiang ;
Gui, Yijie ;
Hu, Weiming ;
Zhu, Jinqing ;
Zeng, Mengqian ;
Li, Yu ;
Xu, Yunbi .
PLOS ONE, 2009, 4 (10)
[8]   Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria [J].
Gasiunas, Giedrius ;
Barrangou, Rodolphe ;
Horvath, Philippe ;
Siksnys, Virginijus .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2012, 109 (39) :E2579-E2586
[9]   Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6 [J].
Gootenberg, Jonathan S. ;
Abudayyeh, Omar O. ;
Kellner, Max J. ;
Joung, Julia ;
Collins, James J. ;
Zhang, Feng .
SCIENCE, 2018, 360 (6387) :439-+
[10]   Nucleic acid detection with CRISPR-Cas13a/C2c2 [J].
Gootenberg, Jonathan S. ;
Abudayyeh, Omar O. ;
Lee, Jeong Wook ;
Essletzbichler, Patrick ;
Dy, Aaron J. ;
Joung, Julia ;
Verdine, Vanessa ;
Donghia, Nina ;
Daringer, Nichole M. ;
Freije, Catherine A. ;
Myhrvold, Cameron ;
Bhattacharyya, Roby P. ;
Livny, Jonathan ;
Regev, Aviv ;
Koonin, Eugene V. ;
Hung, Deborah T. ;
Sabeti, Pardis C. ;
Collins, James J. ;
Zhang, Feng .
SCIENCE, 2017, 356 (6336) :438-+