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.
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•Active expression of multiple CRISPR nucleases and gRNAs in an E. coli TXTL system•Repression activity of dCas9 strongly correlated between TXTL and E. coli•A TXTL-based PAM assay allowed characterization of five Cpf1 nucleases•Determined specificities of 24 anti-CRISPR proteins against five Cas9 nucleases
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.
Precise genome editing of plants has the potential to reshape global agriculture through the targeted engineering of endogenous pathways or the introduction of new traits. To develop a CRISPR ...nuclease-based platform that would enable higher efficiencies of precise gene insertion or replacement, we screened the Cpf1 nucleases from Francisella novicida and Lachnospiraceae bacterium ND2006 for their capability to induce targeted gene insertion via homology directed repair. Both nucleases, in the presence of a guide RNA and repairing DNA template flanked by homology DNA fragments to the target site, were demonstrated to generate precise gene insertions as well as indel mutations at the target site in the rice genome. The frequency of targeted insertion for these Cpf1 nucleases, up to 8%, is higher than most other genome editing nucleases, indicative of its effective enzymatic chemistry. Further refinements and broad adoption of the Cpf1 genome editing technology have the potential to make a dramatic impact on plant biotechnology.
Members of the MscS superfamily of mechanosensitive ion channels function as osmotic safety valves, releasing osmolytes under increased membrane tension. MscS homologs exhibit diverse topology and ...domain structure, and it has been proposed that the more complex members of the family might have novel regulatory mechanisms or molecular functions. Here, we present a study of MscS-Like (MSL)10 from Arabidopsis thaliana that supports these ideas. High-level expression of MSL10-GFP in Arabidopsis induced small stature, hydrogen peroxide accumulation, ectopie cell death, and reactive oxygen species-and cell death-associated gene expression. Phosphomimetic mutations in the MSL10 N-terminal domain prevented these phenotypes. The phosphorylation state of MSL10 also regulated its ability to induce cell death when transiently expressed in Nicotiana benthamiana leaves but did not affect subcellular localization, assembly, or channel behavior. Finally, the N-terminal domain of MSL10 was sufficient to induce cell death in tobacco, independent of phosphorylation state. We conclude that the plant-specific N-terminal domain of MSL10 is capable of inducing cell death, this activity is regulated by phosphorylation, and MSL10 has two separable activities—one as an ion channel and one as an inducer of cell death. These findings further our understanding of the evolution and significance of mechanosensitive lon channels.
Suboptimal vaccine immunogenicity and antigenic mismatch, compounded by poor uptake, means that influenza remains a major global disease. T cells recognizing peptides derived from conserved viral ...proteins could enhance vaccine-induced cross-strain protection.
To investigate the kinetics, phenotypes, and function of influenza virus-specific CD8
resident memory T (Trm) cells in the lower airway and infer the molecular pathways associated with their response to infection
.
Healthy volunteers, aged 18-55, were inoculated intranasally with influenza A/California/4/09(H1N1). Blood, upper airway, and (in a subgroup) lower airway samples were obtained throughout infection. Symptoms were assessed by using self-reported diaries, and the nasal viral load was assessed by using quantitative PCR. T-cell responses were analyzed by using a three-color FluoroSpot assay, flow cytometry with MHC I-peptide tetramers, and RNA sequencing, with candidate markers being confirmed by using the immunohistochemistry results for endobronchial biopsy specimens.
After challenge, 57% of participants became infected. Preexisting influenza-specific CD8
T cells in blood correlated strongly with a reduced viral load, which peaked at Day 3. Influenza-specific CD8
T cells in BAL fluid were highly enriched and predominantly expressed the Trm markers CD69 and CD103. Comparison between preinfection CD8
T cells in BAL fluid and blood by using RNA sequencing revealed 3,928 differentially expressed genes, including all major Trm-cell markers. However, gene set enrichment analysis of BAL-fluid CD8
T cells showed primarily innate cell-related pathways and, during infection, included upregulation of innate chemokines (
,
, and
) that were also expressed by CD8
cells in bronchial tissues.
CD8
Trm cells in the human lung display innate-like gene and protein expression that demonstrates blurred divisions between innate and adaptive immunity. Clinical study registered with www.clinicaltrials.gov (NCT02755948).
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