(734a) Structure-Function Design Principles at Nucleotide Resolution for Crispr Small Guide RNAs

The recent discovery of the Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) system in prokaryotes has triggered a rapid expansion in our ability to edit genomes and control gene expression. In one example, the engineered type II CRISPR system from S. pyogenes, Cas9 (CRISPR associated protein 9) binds to a synthetic small guide RNA (sgRNA) that targets Cas9 to a specific DNA sequence for cleavage [1]. This ability to target Cas9 by only changing part of the sgRNA has led to the widespread use of the Cas9-sgRNA system for genome editing [2]. Further engineering of Cas9 has also converted this system into an RNA-guided platform for sequence-specific control of gene expression [3], making the Cas9-sgRNA system one of the most versatile biotechnology platforms for cellular manipulation currently at our disposal. Thus, there is great interest in understanding the design principles behind the type II CRISPR system to facilitate its further engineering. In this work, we explore the structural requirements of the sgRNA for binding dCas9 (catalytically dead Cas9) and targeting DNA sequences for regulation in E. coli. We find that nucleotide flexibility is critical for function within specific regions of the sgRNA. This work is uncovering new design principles of sgRNAs that will allow further engineering of this system to make new, more sophisticated regulators from the type II CRISPR system.

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2. Sander, J. D., and Joung, J. K. (2014) CRISPR-Cas systems for editing, regulating and targeting genomes. Nat. Biotechnol. 32, 347–355.
3. Qi, L. S., et al. (2013) Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression. Cell 152, 1173–1183.