Single Stage Purification of CRISPR/Cas13a Nuclease via Metal-Chelating Chromatography Following Heterologous Expression
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le Stage Purification of CRISPR/Cas13a Nuclease via Metal-Chelating Chromatography Following Heterologous Expression with the Preservation of Collateral Ribonuclease Activity L. K. Kurbatova, *, S. P. Radkoa, **, S. V. Kravchenkob, O. I. Kiselevaa, N. D. Durmanovb, and A. V. Lisitsaa, b aV.N. b
Orekhovich Institute of Biomedical Chemistry, Moscow, 119121 Russia X-Bio Institute, Tyumen State University, Tyumen, 625003 Russia *e-mail: [email protected] **e-mail: [email protected] Received April 30, 2020; revised June 24, 2020; accepted July 2, 2020
Abstract—CRISPR/Cas13a nucleases are currently considered to be the basis for the development of a new generation of biosensors for the ultrasensitive, in-field detection of bacterial and viral pathogens. A recombinant Cas13a nuclease with functional affinity was obtained as a result of heterologous expression in E. coli with a single-step purification process via metal-chelating chromatography with the N-terminal polyhistidine tag. The simplified procedure of Cas13a nuclease purification broadens the possibilities for the development and practical application of diagnostic biosensing systems based on it. Moreover, our results indicate that the currently uncharacterized protein U2PWF1 of Leptotrichia wadei represents Cas13a nuclease. Keywords: CRISPR-Cas, Cas13a ribonuclease, purification, metal-chelating chromatography, collateral activity DOI: 10.1134/S0003683820060071
INTRODUCTION The nucleases of clustered, regularly interspaced, short palindromic repeats (CRISPR) are an essential component of the molecular systems involving CRISPR and CRISPR-associated proteins (CRISPR-Cas). The CRISPR-Cas system is a bacterial and archaeal immune system that confers resistance to foreign genetic elements, such as those present within plasmids and bacteriophages [1, 2]. The ability of CRISPR-Cas systems to recognize selectively and cleave specific DNA strands is the basis for the most common technology of targeted genome editing, which primarily uses Cas9 nuclease and genetically modified versions of it (CRISPR-Cas9 technology [3]). Target recognition relies on the formation of a complex comprising the Cas nuclease and guide RNA (gRNA). gRNA contains a domain (spacer) complementary to the target DNA domain (protospacer motif) and a motif responsible for Cas-nuclease binding (repeat). Upon the formation of the spacer–protospacer heteroduplex, the Cas-nuclease/gRNA complex cleaves the target DNA if a specific nucleotide motif (protospacer adjacent motif, PAM) is located in close proximity to the protospacer [3]. The CRISPR-nuclease Cas13a is a type-VI CRISPR-Cas system and has several features that dis-
tinguish it from most CRISPR nucleases, including Cas9. Cas13a is a ribonuclease that exerts selective recognition of target RNA (via the formation of a complementary duplex between the gRNA spacer and the specific domain on RNA target), followed by its cleavage independent of PAM [4, 5]. After activation and cleavage of the target RNA, the nuclease loses its specificity (activity only
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