Application Notes

Determination of low-picomolar affinities of sgRNAs and crRNA/tracrRNAs for Cas9

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2 APPLICATION NOTE ©2017 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. systems, as exemplified by CRISPR-Cas9 (Cas9 = CRISPR associated protein 9), are adaptive immune systems used by bacteria and archaea to defend against viral infections. Cas9 is an RNA-guided DNA endonuclease from Streptococcus pyogenes, which utilizes Cas9 to memorize 3 and later interrogate and cleave foreign DNA. 4 The combination of Cas9 from S. pyogenes and a synthetic single-guide RNA (sgRNA) containing the guide region, in addition to a duplex of CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA) has been used as a two-component programmable system for genetic manipulation in various organisms. 5-7 The process of optimizing the CRISPR/Cas9 system for therapeutic approaches requires accurate quantification of the binding affinities of diverse RNA constructs to Cas9. In this Application Note, we demonstrate that MST offers unprecedented advantages over classical methods for DNA/RNA/protein interaction studies, such as immuno-precipitation or electrophoretic mobility shi assay (EMSA). Immunoprecipitation studies are laborious and require the use of antibodies. Classical radioisotope or biotin/ streptavidin-based EMSA approaches require several steps, the use of hazardous material, problematic waste disposal and inconvenient detection. Additionally, a chemical equilibrium cannot be reached in an EMSA assay, and RNA dissociation from protein during electrophoresis can prevent detection of an interaction. The MST assay offers excellent sensitivity with minute amounts of non- hazardous fluorescent-labeled oligonucleotides or proteins, the protein/RNA complexes are detected Introduction CRISPR/Cas9 genome editing technologies are one of the most significant discoveries of this decade with a vast potential in human therapeutics, food science and other fields. Recently, the US Department of Agriculture approved the cultivation and commercialization of CRISPR/Cas9 gene-edited button mushroom (Agaricus bisporus). 1 Furthermore, an advisory committee at the US National Institutes of Health (NIH) approved a proposal to use CRISPR-Cas9 to help augment cancer therapies that rely on enlisting a patient´s T cells. Clustered regularly interspaced short palindromic repeats (CRISPRs) are segments of prokaryotic DNA with short repeating segments. The CRISPR-Cas Figure 1. Crystal structure of Streptococcus pyogenes Cas9 in complex with guide RNA (blue) and target DNA (red) (PDB ID: 4OO8) 2

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