A New Toolkit for Evaluating Gene Functions using Conditional Cas9 Stabilization

被引:0
|
作者
Tepes, Polona Safaric [1 ,2 ]
Sordella, Raffaella [1 ]
机构
[1] Cold Spring Harbor Lab, Cold Spring Harbor, NY 11724 USA
[2] Univ Ljubljana, Fac Pharm, Ljubljana, Slovenia
来源
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS | 2021年 / 175期
关键词
D O I
10.3791/60685
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
The clustered regularly interspaced short palindromic repeat- (CRISPR-) associated protein 9 (CRISPR/Cas9) technology has become a prevalent laboratory tool to introduce accurate and targeted modifications in the genome. Its enormous popularity and rapid spread are attributed to its easy use and accuracy compared to its predecessors. Yet, the constitutive activation of the system has limited applications. In this paper, we describe a new method that allows temporal control of CRISPR/Cas9 activity based on conditional stabilization of the Cas9 protein. Fusing an engineered mutant of the rapamycin-binding protein FKBP12 to Cas9 (DD-Cas9) enables the rapid degradation of Cas9 that in turn can be stabilized by the presence of an FKBP12 synthetic ligand (Shield-1). Unlike other inducible methods, this system can be adapted easily to generate bi-cistronic systems to co-express DD-Cas9 with another gene of interest, without conditional regulation of the second gene. This method enables the generation of traceable systems as well as the parallel, independent manipulation of alleles targeted by Cas9 nuclease. The platform of this method can be used for the systematic identification and characterization of essential genes and the interrogation of the functional interactions of genes in in vitro and in vivo settings.
引用
收藏
页数:15
相关论文
共 50 条
  • [41] Gene insertion in Saccharomyces cerevisiae using the CRISPR/Cas9 system
    Xuan Guo
    Yuehua Wang
    Meixiao Wu
    Jianbing Hu
    Xuefei Wang
    Ming Yu
    Hui Tang
    3 Biotech, 2021, 11
  • [42] Tissue-specific gene targeting using CRISPR/Cas9
    Ablain, J.
    Zon, L. I.
    ZEBRAFISH: GENETICS, GENOMICS, AND TRANSCRIPTOMICS, 4TH EDITION, 2016, 135 : 189 - 202
  • [43] Targeted gene disruption in Xenopus laevis using CRISPR/Cas9
    Wang, Fengqin
    Shi, Zhaoying
    Cui, Yan
    Guo, Xiaogang
    Shi, Yun-Bo
    Chen, Yonglong
    CELL AND BIOSCIENCE, 2015, 5
  • [44] A CRISPR Cas9 high-throughput genome editing toolkit for kinetoplastids
    Beneke, Tom
    Madden, Ross
    Makin, Laura
    Valli, Jessica
    Sunter, Jack
    Gluenz, Eva
    ROYAL SOCIETY OPEN SCIENCE, 2017, 4 (05): : 1 - 16
  • [45] Using Cas9 electroporation and AAV templates to generate knock-in and conditional alleles in mice
    Gertsenstein, Marina
    Lintott, Lauri G.
    Laurin, Valerie
    Chan, Linda
    Fan-Lan, Qing
    Tondat, Sandra
    Patel, Amit
    Cruz, Maribelle
    Nutter, Lauryl M. J.
    TRANSGENIC RESEARCH, 2023, 32 : S11 - S11
  • [46] A DNA assembly toolkit to unlock the CRISPR/Cas9 potential for metabolic engineering
    Tigran V. Yuzbashev
    Evgeniya Y. Yuzbasheva
    Olga E. Melkina
    Davina Patel
    Dmitrii  Bubnov
    Heiko Dietz
    Rodrigo Ledesma-Amaro
    Communications Biology, 6
  • [47] Conditional targeting of Ispd using paired Cas9 nickase and a single DNA template in mice
    Lee, Angus Yiu-fai
    Lloyd, Kevin C. Kent
    FEBS OPEN BIO, 2014, 4 : 637 - 642
  • [48] A Programmable CRISPR/Cas9 Toolkit Improves Lycopene Production in Bacillus subtilis
    Liu, Yang
    Cheng, Haijiao
    Li, Haoni
    Zhang, Yingzhe
    Wang, Meng
    APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2023, 89 (06)
  • [49] A DNA assembly toolkit to unlock the CRISPR/Cas9 potential for metabolic engineering
    Yuzbashev, Tigran V.
    Yuzbasheva, Evgeniya Y.
    Melkina, Olga E.
    Patel, Davina
    Bubnov, Dmitrii
    Dietz, Heiko
    Ledesma-Amaro, Rodrigo
    COMMUNICATIONS BIOLOGY, 2023, 6 (01)
  • [50] Cas9-triggered chain ablation of cas9 as a gene drive brake
    Bing Wu
    Liqun Luo
    Xiaojing J Gao
    Nature Biotechnology, 2016, 34 : 137 - 138