Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage

Jiang, F., Taylor, D. W., Chen, J. S., Kornfeld, J. E., Zhou, K., Thompson,A. J., Nogales, E., and Doudna, J. A., et al.

Science 351, 867-871 (2016)

Speaker: Tsai-Yu Wu (吳彩瑜)                                             Time: 14:00~15:00, Apr. 27, 2016

Commentator: Prof. Woei-Jer Chuang (莊偉哲老師)          Place: Room 601

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) are defense system present in many bacteria and archaea. The CRISPER-Cas systems provide adaptive immunity to detect and eliminate invading genetic elements¹. They are classified into three types based on the DNA sequences. The type II CRISPR mechanism is unique because only one Cas protein (Cas9) is required for gene degradation². In the type II CRISPR/Cas system, a segment of CRISPR RNA (crRNA) forms a DNA-RNA hybrid helix with invading dsDNA. As crRNA forms a hybrid helix with the complementary strand on dsDNA (target DNA), the other strand of the dsDNA (non-target strand) is displaced. Moreover, crRNA and a trans-activating crRNA (tracrRNA) or an engineered single-guide RNA (sgRNA) interacts with Cas9 endonuclease to form the R-loop which regulates DNA degradation³. However, in all available structures, the catalytic sites in HNH and RuvC domains of Cas9 endonucleases are not positioned properly for target-strand DNA cleavage site due to lack of an intact non-target strand in these complex. Furthermore, the R-loop configuration for positioning each DNA strand to be cleaved by Cas9 nuclease domains is unclear. To understand the actual Cas9 conformational change during cleavage-competent state, the authors solved the crystal structure of Streptococcus pyogenes Cas9-sgRNA-dsDNA complex. This structure reveals that the non-target DNA strand located near the RuvC domain and the unwinding of double-stranded DNA induces conformational changes in Cas9 HNH and RuvC domain. In addition, the authors unraveled that Cas9 holds both ends of unwound dsDNA resulting in creating the bending of the DNA double helix for R-loop formation by cryo-electron microscopy and crystallography. Moreover, the distorted DNA double helix traverses the Cas9 and contacts with the nuclease domain. In conclusion, this paper provides the structural basis of the R-loop formation essential for the molecular mechanism of CRISPR/Cas9.

References

1.         van der Oost, J., et al. Unravelling the structural and mechanistic basis of CRISPR-Cas systems. Nature reviews. Microbiology 12, 479-492 (2014).

2.         Deltcheva, E., et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 471, 602-607 (2011).

3.         Szczelkun, M.D., et al. Direct observation of R-loop formation by single RNA-guided Cas9 and Cascade effector complexes. Proceedings of the National Academy of Sciences of the United States of America 111, 9798-9803 (2014).