• Rapid Testing of CRISPR Nucleases and Guide RNAs in an Cell-Free Transcription-Translation System.

      Marshall, Ryan; Beisel, Chase L; Noireaux, Vincent; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Elsevier (CellPress), 2020-06-03)
      We present a protocol to rapidly test DNA binding and cleavage activity by CRISPR nucleases using cell-free transcription-translation (TXTL). Nuclease activity is assessed by adding DNA encoding a nuclease, a guide RNA, and a targeted reporter to a TXTL reaction and by measuring the fluorescence for several h. The reactions, performed in a few microliters, allow for parallel testing of many nucleases and guide RNAs. The protocol includes representative results for (d)Cas9 from Streptococcus pyogenes targeting a GFP reporter gene. For complete information on the generation and use of this protocol, please refer to the paper by Marshall et al. (2018).
    • Sequence-independent RNA sensing and DNA targeting by a split domain CRISPR-Cas12a gRNA switch.

      Collins, Scott P; Rostain, William; Liao, Chunyu; Beisel, Chase L; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Oxgord Uiversity Press, 2021-02-22)
      CRISPR technologies increasingly require spatiotemporal and dosage control of nuclease activity. One promising strategy involves linking nuclease activity to a cell's transcriptional state by engineering guide RNAs (gRNAs) to function only after complexing with a 'trigger' RNA. However, standard gRNA switch designs do not allow independent selection of trigger and guide sequences, limiting gRNA switch application. Here, we demonstrate the modular design of Cas12a gRNA switches that decouples selection of these sequences. The 5' end of the Cas12a gRNA is fused to two distinct and non-overlapping domains: one base pairs with the gRNA repeat, blocking formation of a hairpin required for Cas12a recognition; the other hybridizes to the RNA trigger, stimulating refolding of the gRNA repeat and subsequent gRNA-dependent Cas12a activity. Using a cell-free transcription-translation system and Escherichia coli, we show that designed gRNA switches can respond to different triggers and target different DNA sequences. Modulating the length and composition of the sensory domain altered gRNA switch performance. Finally, gRNA switches could be designed to sense endogenous RNAs expressed only under specific growth conditions, rendering Cas12a targeting activity dependent on cellular metabolism and stress. Our design framework thus further enables tethering of CRISPR activities to cellular states.
    • Tunable self-cleaving ribozymes for modulating gene expression in eukaryotic systems.

      Jacobsen, Thomas; Yi, Gloria; Al Asafen, Hadel; Jermusyk, Ashley A; Beisel, Chase L; Reeves, Gregory T; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (PLOS, 2020-04-30)
      Advancements in the field of synthetic biology have been possible due to the development of genetic tools that are able to regulate gene expression. However, the current toolbox of gene regulatory tools for eukaryotic systems have been outpaced by those developed for simple, single-celled systems. Here, we engineered a set of gene regulatory tools by combining self-cleaving ribozymes with various upstream competing sequences that were designed to disrupt ribozyme self-cleavage. As a proof-of-concept, we were able to modulate GFP expression in mammalian cells, and then showed the feasibility of these tools in Drosophila embryos. For each system, the fold-reduction of gene expression was influenced by the location of the self-cleaving ribozyme/upstream competing sequence (i.e. 5' vs. 3' untranslated region) and the competing sequence used. Together, this work provides a set of genetic tools that can be used to tune gene expression across various eukaryotic systems.
    • Your Base Editor Might Be Flirting with Single (Stranded) DNA: Faithful On-Target CRISPR Base Editing without Promiscuous Deamination.

      Collins, Scott P; Beisel, Chase L; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Elsevier, 2020-09-03)
      Jin et al. (2020) engineered new variants of CRISPR base editors that make precise genomic edits in rice protoplasts while minimizing untargeted mutagenesis.