Rapid and Scalable Characterization of CRISPR Technologies Using an E. coli Cell-Free Transcription-Translation System

Abstract

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.

Distinct timescales of RNA regulators enable the construction of a genetic pulse generator

“…P y tot is the total amount of reporter promoters, M i is the translationally initialized mRNA, and G is the immature GFP protein. We note that no protein degradation rate is included because proteins do not degrade in TXTL unless degradation tags are included, (Garamella et al, ) and there is no translation rate for dCas9 because extracts were made from E. coli cells expressing dCas9 (Marshall et al, ).…”

Multiplex transcriptional characterizations across diverse bacterial species using cell‐free systems

“…By shortcutting time-consuming cloning and transformation steps, TXTL reactions can directly yield proteins and metabolites straight from DNA that encodes biosynthetic genes, operons, or pathways. TXTL has also been useful for characterizing synthetic gene circuits with multi-layered components and dynamic behaviors (Takahashi et al, 2015;Borkowski et al, 2018;Marshall et al, 2018). However, the correspondence between in vitro measurements and actual in vivo conditions in live cells remains understudied (Jewett et al, 2008;Chappell et al, 2013;Siegal-Gaskins et al, 2014).…”

Cell‐free protein synthesis: The transition from batch reactions to minimal cells and microfluidic devices

“…Notably, genomic material withdrawal from chassis organism bestows circumstances in which desired synthesis reactions are achievable at high rates . Thus, such features enable prevailing utilization of CFPS systems, which are also complementary to in vivo expression for various applications such as biocatalyst development (Rolf, Rosenthal, & Lütz, 2019), complex product fabrication (Chi, Wang, Li, Ren, & Huang, 2015), disease detection (Soltani, Davis, Ford, Nelson, & Bundy, 2018), glycoprotein synthesis (Jaroentomeechai et al, 2018), prototyping minimal cells (Yue, Zhu, & Kai, 2019), synthetic gene networks (Dubuc et al, 2019), as well as protein engineering (Hong, Kwon, & Jewett, 2014;Venkat, Chen, Gan, & Fan, Sheng, Lei, Yuan and Feng (2017) Functional CRISPR gene editing toolkit Marshall et al (2018) Pathway/network prototyping UDP-N-acetylglucosamine and UDP-GlcNAc pathway Zhou et al (2010) CRISPR-mediated gene activation and repression of the reporter and endogenous genes in mammalian cells Nakamura et al (2019) Protein engineering E. coli strain mutant for release factor 1 allows turning UAG termination codon into a sense codon for site-specific incorporation of nonnatural amino acids into proteins for selective conjugation with different biomolecules Adachi et al (2019) Incorporation of uAA AzF allows for site-specific mono and dipegylation of T4 Lysozyme Wilding et al (2018) Incorporating optimized nonnatural amino acids site-specifically, the feasibility of conjugating a DBCO-PEG-monomethyl auristatin (DBCO-PEG-MMAF) drug by para-azidomethyl-L-phenylalanine (pAMF) to the tumor-specific, Her2-binding IgG Trastuzumab Zimmerman et al (2014) Biosensing Cheomogenic detection of estrogenic endocrine disruptors (hERb) in human blood and urine Salehi et al (2018) Glycoengineering Site-specific controlled glycosylation of proteins (glycoproteins) using E. coli extracts enriched for oligosaccharyltransferases and lipidlinked oligosaccharides Jaroentomeechai et al (2018) Site-directed incorporation of AzF, as well as ppropargyloxyphenylalanine from Sf21 insect cell, extracts for engineered O-glycosylation site of EPO Zemella et al (2018) Protein evolution: ribosome display Protein-tRNA-ribosome-mRNA complex for affinity selection of the nascent peptides on an immobilized monoclonal antibody specific for the peptide dynorphin Mattheakis, Bhatt and Dower (1994) Protein evolution: mRNA display Protein-puromycin-mRNA complex Roberts...…”