Mapping the sugar dependency for rational generation of a DNA-RNA hybrid-guided Cas9 endonuclease

The CRISPR–Cas9 RNA-guided endonuclease system allows precise and efficient modification of complex genomes and is continuously developed to enhance specificity, alter targeting and add new functional moieties. However, one area yet to be explored is the base chemistry of the associated RNA molecules. Here we show the design and optimisation of hybrid DNA–RNA CRISPR and tracr molecules based on structure-guided approaches. Through careful mapping of the ribose requirements of Cas9, we develop hybrid versions possessing minimal RNA residues, which are sufficient to direct specific nuclease activity in vitro and in vivo with reduced off-target activity. We identify critical regions within these molecules that require ribose nucleotides and show a direct correlation between binding affinity/stability and cellular activity. This is the first demonstration of a non-RNA-guided Cas9 endonuclease and first step towards eliminating the ribose dependency of Cas9 to develop a XNA-programmable endonuclease.CRISPR-Cas9 systems are being continually improved to enhance specificity and improve functionality. Here the authors design hybrid DNA-RNA guide and tracr molecules to direct Cas9 nuclease activity with reduced off-target effects.

[1]  Samuel H Sternberg,et al.  Rational design of a split-Cas9 enzyme complex , 2015, Proceedings of the National Academy of Sciences.

[2]  Hiroshi Nishimasu.  Crystal Structure of Cas9 , 2015 .

[3]  J. Keith Joung,et al.  731. High-Fidelity CRISPR-Cas9 Nucleases with No Detectable Genome-Wide Off-Target Effects , 2016 .

[4]  N. Kouprina,et al.  Highly efficient CRISPR/Cas9-mediated TAR cloning of genes and chromosomal loci from complex genomes in yeast , 2015, Nucleic acids research.

[5]  Michel Sadelain,et al.  Safe harbours for the integration of new DNA in the human genome , 2011, Nature Reviews Cancer.

[6]  J. Doudna,et al.  A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity , 2012, Science.

[7]  D. Rueda,et al.  Activation-induced deoxycytidine deaminase (AID) co-transcriptional scanning at single-molecule resolution , 2015, Nature Communications.

[8]  CD Richardson,et al.  Non-homologous DNA increases gene disruption efficiency by altering DNA repair outcomes , 2016, Nature Communications.

[9]  Wenjun Jiang,et al.  Cas9-Assisted Targeting of CHromosome segments CATCH enables one-step targeted cloning of large gene clusters , 2015, Nature Communications.

[10]  J. Keith Joung,et al.  Improving CRISPR-Cas nuclease specificity using truncated guide RNAs , 2014, Nature Biotechnology.

[11]  U. Rant,et al.  Polymerase/DNA interactions and enzymatic activity: multi-parameter analysis with electro-switchable biosurfaces , 2015, Scientific Reports.

[12]  Martin J. Aryee,et al.  Engineered CRISPR-Cas9 nucleases with altered PAM specificities , 2015, Nature.

[13]  D. Rueda,et al.  RNA folding dynamics by single-molecule fluorescence resonance energy transfer. , 2009, Methods.

[14]  Yuya Ito,et al.  Corrigendum: Mutations in CDCA7 and HELLS cause immunodeficiency–centromeric instability–facial anomalies syndrome , 2016, Nature Communications.

[15]  D. Herschlag,et al.  Lessons from Enzyme Kinetics Reveal Specificity Principles for RNA-Guided Nucleases in RNA Interference and CRISPR-Based Genome Editing. , 2017, Cell systems.

[16]  Jennifer A. Doudna,et al.  DNA interrogation by the CRISPR RNA-guided endonuclease Cas9 , 2014, Nature.

[17]  N. Walter,et al.  siRNA-Like Double-Stranded RNAs Are Specifically Protected Against Degradation in Human Cell Extract , 2011, PloS one.

[18]  Jin-Soo Kim,et al.  Structural roles of guide RNAs in the nuclease activity of Cas9 endonuclease , 2016, Nature Communications.

[19]  Israel Steinfeld,et al.  Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells , 2015, Nature Biotechnology.

[20]  J. Doudna,et al.  The new frontier of genome engineering with CRISPR-Cas9 , 2014, Science.

[21]  B. van Steensel,et al.  Easy quantitative assessment of genome editing by sequence trace decomposition , 2014, Nucleic acids research.

[22]  Howard M. Salis,et al.  A Biophysical Model of CRISPR/Cas9 Activity for Rational Design of Genome Editing and Gene Regulation , 2016, PLoS Comput. Biol..

[23]  Yilong Li,et al.  Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library , 2013, Nature Biotechnology.

[24]  James E. DiCarlo,et al.  RNA-Guided Human Genome Engineering via Cas9 , 2013, Science.

[25]  Noriyuki Kurita,et al.  Hybridization energies of double strands composed of DNA, RNA, PNA and LNA. , 2007, Chemical physics letters.

[26]  Todd E. Scheetz,et al.  Somatic Mutagenesis with a Sleeping Beauty Transposon System Leads to Solid Tumor Formation in Zebrafish , 2011, PloS one.

[27]  Real-time observation of DNA recognition and rejection by the RNA-guided endonuclease Cas9 , 2016, bioRxiv.

[28]  E. Lesnik,et al.  Relative thermodynamic stability of DNA, RNA, and DNA:RNA hybrid duplexes: relationship with base composition and structure. , 1995, Biochemistry.

[29]  Jennifer A. Doudna,et al.  Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage , 2016, Science.

[30]  Jennifer A. Doudna,et al.  Conformational control of DNA target cleavage by CRISPR–Cas9 , 2015, Nature.

[31]  David A. Scott,et al.  Rationally engineered Cas9 nucleases with improved specificity , 2015, Science.

[32]  Jacob E Corn,et al.  Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA , 2016, Nature Biotechnology.

[33]  David Rueda,et al.  Single-molecule FRET of protein-nucleic acid and protein-protein complexes: surface passivation and immobilization. , 2010, Methods.

[34]  Huimin Zhao,et al.  Programmable DNA-Guided Artificial Restriction Enzymes. , 2017, ACS synthetic biology.