Rapid and Scalable Characterization of CRISPR Technologies Using an E. coli Cell-Free Transcription-Translation System.
Rapid and Scalable Characterization of CRISPR Technologies Using an E. coli Cell-Free Transcription-Translation System.
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DOI：
10.1016/j.molcel.2017.12.007
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发表时间：
2018-01-04
期刊：
影响因子：
16
通讯作者：
Noireaux V
中科院分区：
文献类型：
--
作者：
Marshall R;Maxwell CS;Collins SP;Jacobsen T;Luo ML;Begemann MB;Gray BN;January E;Singer A;He Y;Beisel CL;Noireaux V
CRISPR-Cas systems offer versatile technologies for genome engineering, yet their implementation has been outpaced by ongoing discoveries of new Cas nucleases and anti-CRISPR proteins. Here, we present the use of E. coli cell-free transcription-translation (TXTL) systems to vastly improve the speed and scalability of CRISPR characterization and validation. TXTL can express active CRISPR machinery from added plasmids and linear DNA, and TXTL can output quantitative dynamics of DNA cleavage and gene repression—all without protein purification or live cells. We used TXTL to measure the dynamics of DNA cleavage and gene repression for single- and multi-effector CRISPR nucleases, predict gene repression strength in E. coli, determine the specificities of 24 diverse anti-CRISPR proteins, and develop a fast and scalable screen for protospacer-adjacent motifs that was successfully applied to five uncharacterized Cpf1 nucleases. These examples underscore how TXTL can facilitate the characterization and application of CRISPR technologies across their many uses. In Brief Marshall et al. demonstrate that an E. coli cell-free transcription-translation (TXTL) system can be used to improve the speed and scalability of characterizing CRISPR nucleases and their accessory factors. The method will facilitate the discovery of uncharacterized CRISPR nucleases and anti-CRISPR proteins and aid the validation of designed gRNAs.
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影响因子：
64.8
作者：
Bondy-Denomy J;Garcia B;Strum S;Du M;Rollins MF;Hidalgo-Reyes Y;Wiedenheft B;Maxwell KL;Davidson AR
通讯作者：
Davidson AR
DOI：
10.1126/science.aaf5573
发表时间：
2016-08-05
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Abudayyeh OO;Gootenberg JS;Konermann S;Joung J;Slaymaker IM;Cox DB;Shmakov S;Makarova KS;Semenova E;Minakhin L;Severinov K;Regev A;Lander ES;Koonin EV;Zhang F
通讯作者：
Zhang F
影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle
影响因子：
20.3
作者：
Kanter, Gregory;Yang, Junhao;Levy, Ronald
通讯作者：
Levy, Ronald
影响因子：
4.6
作者：
Begemann MB;Gray BN;January E;Gordon GC;He Y;Liu H;Wu X;Brutnell TP;Mockler TC;Oufattole M
通讯作者：
Oufattole M