Publications

Mageeney, Catherine M.; Schoeniger, Joseph S.; Williams, Kelly P.

Abstract not provided.

Mosesso, Richard A.; Courtney, Colleen M.; Collette, Nicole C.; Negrete, Oscar N.; Schoeniger, Joseph S.

Abstract not provided.

Schoeniger, Joseph S.

Abstract not provided.

Mageeney, Catherine M.; Williams, Kelly P.; Schoeniger, Joseph S.

Abstract not provided.

Saada, Edwin A.; Schoeniger, Joseph S.; Negrete, Oscar N.

Abstract not provided.

Harmon, Brooke N.; Light, Yooli K.; Tran-Gyamfi, Mary B.; Schoeniger, Joseph S.; Murthy, Niren M.; Han, Hesong H.

Abstract not provided.

Branda, Steven B.; Wang, Pei-Li W.; LaBauve, Annette E.; Sinha, Anupama S.; Poorey, Kunal N.; Williams, Kelly P.; Michailidis, George M.; Schoeniger, Joseph S.; Mageeney, Catherine M.; Courtney, Colleen M.; El-Etr, Sahar E.; Franco, Magda F.; Lao, Victoria L.; D'haeseleer, Patrik D.; Pena, Jose P.; Segelke, Brent S.

Abstract not provided.

Courtney, Colleen M.; Johnston, Robert K.; LaBauve, Annette E.; Collete, Nicole C.; Edwin, Saada A.; Negrete, Oscar N.; Branda, Steven B.; Butler, Kimberly B.; Schoeniger, Joseph S.

Abstract not provided.

LaBauve, Annette E.; Saada, Edwin A.; Brinker, Jeff B.; Schoeniger, Joseph S.; Negrete, Oscar N.

Abstract not provided.

Schoeniger, Joseph S.; Negrete, Oscar N.

Abstract not provided.

Analytical Methods

Phaneuf, Christopher R.; Seamon, Kyle J.; Eckles, Tyler P.; Sinha, Anchal; Schoeniger, Joseph S.; Harmon, Brooke N.; Meagher, Robert M.; Abhyankar, Vinay V.; Koh, Chung-Yan K.

The discovery of the RNA-guided DNA nuclease CRISPR-Cas9 has enabled the targeted editing of genomes from diverse organisms, but the permanent and inheritable nature of genome modification also poses immense risks. The potential for accidental exposure, malicious use, or undesirable persistence of Cas9 therapeutics and off-target genome effects highlight the need for detection assays. Here we report a centrifugal microfluidic platform for the measurement of both Cas9 protein levels and nuclease activity. Because Cas9 from many bacterial species have been adapted for biotechnology applications, we developed the capability to detect Cas9 from the widely-used S. pyogenes, as well as S. aureus, N. meningitidis, and S. thermophilus using commercially-available antibodies. Further, we show that the phage-derived anti-CRISPR protein AcrIIC1, which binds to Cas9 from several species, can be used as a capture reagent to broaden the species range of detection. As genome modification generally requires Cas9 nuclease activity, a fluorescence-based sedimentation nuclease assay was also incorporated to allow the sensitive and simultaneous measurement of both Cas9 protein and activity in a single biological sample.

Phaneuf, Christopher P.; Seamon, Kyle J.; Eckles, Tyler P.; Sinha, Anchal S.; Schoeniger, Joseph S.; Harmon, Brooke N.; Meagher, Robert M.; Abhyankar, Vinay A.; Koh, Chung-Yan K.

Abstract not provided.

Scientific Reports

Krishnakumar, Raga K.; Sinha, Anupama S.; Bird, Sara W.; Jayamohan, Harikrishnan; Edwards, Harrison S.; Schoeniger, Joseph S.; Patel, Kamlesh P.; Branda, Steven B.; Bartsch, Michael B.

Emerging sequencing technologies are allowing us to characterize environmental, clinical and laboratory samples with increasing speed and detail, including real-time analysis and interpretation of data. One example of this is being able to rapidly and accurately detect a wide range of pathogenic organisms, both in the clinic and the field. Genomes can have radically different GC content however, such that accurate sequence analysis can be challenging depending upon the technology used. Here, we have characterized the performance of the Oxford MinION nanopore sequencer for detection and evaluation of organisms with a range of genomic nucleotide bias. We have diagnosed the quality of base-calling across individual reads and discovered that the position within the read affects base-calling and quality scores. Finally, we have evaluated the performance of the current state-of-the-art neural network-based MinION basecaller, characterizing its behavior with respect to systemic errors as well as context- and sequence-specific errors. Overall, we present a detailed characterization the capabilities of the MinION in terms of generating high-accuracy sequence data from genomes with a wide range of nucleotide content. This study provides a framework for designing the appropriate experiments that are the likely to lead to accurate and rapid field-forward diagnostics.

Bartsch, Michael B.; Bird, Sara W.; Branda, Steven B.; Edwards, Harrison E.; Jayamohan, Harikrishnan J.; Krishnakumar, Raga K.; Patel, Kamlesh P.; Schoeniger, Joseph S.; Sinha, Anupama S.

Funded through the IHNS/E&HS investment area for FY16-18, the RAPIER LDRD sought to evaluate the potential benefits and applicability of the new Oxford MinION nanopore sequencer to pathogen diagnostic applications in biodefense, biosurveillance, and global/public health. The project had four primary objectives: 1) to investigate the performance of the MinION sequencer while building facility with its operation, 2) to develop microfluidic library prep automation facilitating the use of the MinION in field-forward or point-of-care applications, 3) to leverage CRISPR/Cas9 technology to enable targeted identification of bacterial pathogens, and 4) to capitalize on the real- time data output capabilities of the MinION to enable rapid sequence-based diagnostics. While the rapid evolution of the MinION sequencing technology during the course of the project posed a number of challenges and required a reassessment of initial project priorities, it also provided unique opportunities, notably culminating in our development of the RUBRIC real-time selective sequencing software.

Branda, Steven B.; Courtney, Colleen M.; Sinha, Anupama S.; Poorey, Kunal N.; Schoeniger, Joseph S.; LaBauve, Annette E.; Williams, Kelly P.

Abstract not provided.

Negrete, Oscar N.; Saada, Edwin A.; Strutt, Steven S.; Brinker, C.J.; Schoeniger, Joseph S.

Abstract not provided.

Analytical Chemistry

Seamon, Kyle J.; Light, Yooli K.; Saada, Edwin A.; Schoeniger, Joseph S.; Harmon, Brooke N.

The RNA-guided DNA nuclease Cas9 is now widely used for the targeted modification of genomes of human cells and various organisms. Despite the extensive use of Clustered Regularly Interspaced Palindromic Repeats (CRISPR) systems for genome engineering and the rapid discovery and engineering of new CRISPR-associated nucleases, there are no high-throughput assays for measuring enzymatic activity. The current laboratory and future therapeutic uses of CRISPR technology have a significant risk of accidental exposure or clinical off-target effects, underscoring the need for therapeutically effective inhibitors of Cas9. Here, we develop a fluorescence assay for monitoring Cas9 nuclease activity and demonstrate its utility with S. pyogenes (Spy), S. aureus (Sau), and C. jejuni (Cje) Cas9. The assay was validated by quantitatively profiling the species specificity of published anti-CRISPR (Acr) proteins, confirming the reported inhibition of Spy Cas9 by AcrIIA4 and Cje Cas9 by AcrIIC1 and no inhibition of Sau Cas9 by either anti-CRISPR. To identify drug-like inhibitors, we performed a screen of 189 606 small molecules for inhibition of Spy Cas9. Of 437 hits (0.2% hit rate), six were confirmed as Cas9 inhibitors in a direct gel electrophoresis secondary assay. The high-throughput nature of this assay makes it broadly applicable for the discovery of additional Cas9 inhibitors or the characterization of Cas9 enzyme variants.

Negrete, Oscar N.; Saada, Edwin A.; Brinker, C.J.; Schoeniger, Joseph S.

Abstract not provided.

Bartsch, Michael B.; Krishnakumar, Raga K.; Sinha, Anupama S.; Schoeniger, Joseph S.; Branda, Steven B.; Patel, Kamlesh P.; Edwards, Harrison E.; Bird, Sara W.; Jayamohan, Harikrishnan J.

Abstract not provided.

Krishnakumar, Raga K.; Bartsch, Michael B.; Patel, Kamlesh P.; Branda, Steven B.; Schoeniger, Joseph S.; Edwards, Harrison E.; Bird, Sara W.; Jayamohan, Harikrishnan J.

Abstract not provided.

Lau, Britney L.; Krishnakumar, Raga K.; Wagner, Julian W.; Sinha, Anupama S.; Hudson, Corey H.; Schoeniger, Joseph S.; Branda, Steven B.; Williams, Kelly P.

Abstract not provided.

Sinha, Sankalp S.; Krishnakumar, Raga K.; Williams, Kelly P.; Schoeniger, Joseph S.

Abstract not provided.

Williams, Kelly P.; Lau, Britney Y.; Krishnakumar, Raga K.; Wagner, Julian W.; Hudson, Corey H.; Schoeniger, Joseph S.

Abstract not provided.

Williams, Kelly P.; Krishnakumar, Raga K.; Wagner, Julian W.; Hudson, Corey H.; Schoeniger, Joseph S.; Williams, Kelly P.

Abstract not provided.

Jayamohan, Harikrishnan J.; Bartsch, Michael B.; Krishnakumar, Raga K.; Patel, Kamlesh P.; Branda, Steven B.; Sinha, Anupama S.; Edwards, Harrison E.; Throckmorton, Daniel J.; Bird, Sara W.; Schoeniger, Joseph S.

Abstract not provided.