Reported by: YAN Mingxing
Translated by: QIN Jialu
Edited by: Daniel Penistone
On April 21, Professor Huang Zhiwei’s team from the School of Life Science and Technology published online a research paper in the journal Nature titled The crystal structure of Cpf1 in complex with CRISPR RNA. This study by means of structural biology and biochemistry reveals the crRNA recognition mechanism and the processing of the precursor crRNAs mature molecular mechanism, which has an important scientific significance on recognizing the molecular mechanism of how bacteria defend against viral infection through CRISPR systems. At the same time, this study provides the structural basis for the successful systematic improvement of Cpf1 system which could help the Cpf1 system become the specific and efficient new gene editing system. People can more efficiently “close”, “recover” and “switch” the target gene through precision “surgery”; therefore, making it be possible to combat diseases like cancer and AIDS.
CRISPR-Cas systems are adaptive immune systems by bacterial coding, which through the RNA-guided effector protein cutting the bacteria’s DNA or RNA to defend against viral infection. CRISPR-Cas9 system, as one of the CRISPR-Cas systems, is used as the gene editing tool for the target DNA’s cutting, activation, expression, modification and mutation in the cells. As a powerful tool in research and medical fields, CRISPR-Cas9 system can efficiently and easily “edit” any gene in living cells and has been widely used in biological and medical laboratories worldwide.
The just-discovered CRISPR-Cpf1 system is a new class CRISPR-Cas system, it is able to cut the target DNA substrate in human cells under the crRNA’s guidance. Furthermore, Cpf1 itself is a sequence-specific RNase, which is the only discovered nuclease that has both nuclease sequence specificity and the viability of DNase and RNase. There are some big differences between Cpf1 and Cas9, for example, Cpf1 only needs one copy of crRNA while Cas9 needs longer tracrRNA and crRNA sequences to recognize and cut substrate DNA, and shorter crRNA is much more efficient in the process of cell transfection; the PAM for Cpf1 and Cas9 recognizing the substrate DNA are different; Cpf1 uses sticky ends to cut substrates while Cas9 uses ends, the sticky ends are more conducive to repair the gene after editing.
In this study, Huang’s group first solved the crystal structure of Cpf1 in complex with a crRNA. Surprisingly, the structure of Cpf1 is not as people predicted, but a triangle-shaped architecture with a large positively charged channel at the center. crRNA forms a highly distorted conformation closely integrated with the Cpf1 nuclease domains as a hairpin motif structure, and the end of crRNA 3' matched with the substrate DNA is on the other end of the Cpf1’s channel. Obviously different to the sgRNA matched with Cas9, the guide sequence of crRNA where Cpf1 matched with has no electron density, indicating that this part of the sequence is loosely coupling with the Cpf1 under the condition of no substrate combination. According to Huang Zhiwei’s introduction, observation of the structure shows that one (Mg(H2O)6)2+ ion tightly integrated with the crRNA is critical to stabilize the conformation of crRNA and stimulate the activation of Cpf1. Certainly, we cannot rule out the magnesium ions also directly involved in the catalytic reaction of the substrate. By a comparison between the compound structure of Cpf1 and Cas9, they found that the LHD domain may be the PAM domain which double-stranded DNA substrates combine with.
This study finds that Cpf1 is in a loose conformation with the combination of crRNA, their combination can cause the Cpf1 have obvious conformational change. Very different to the combination between Cas9 and sgRNA, only the repeat sequence of crRNA could be able to cause great change in the Cpf1’s conformation. This consequence shows that the recognition mechanism of this class of short crRNA has huge differences with the long sgRNA combined with Cas9. This structure is on the same surface with the nitrogen atom on the catalytic residues side chain of the H843, K852 and K869, and this structure can form hydrogen bonds with the phosphate group of RNA A(+20). This structure indicates that Cpf1 cutting pre-crRNA to become crRNA is a base catalytic reaction.
Huang Zhiwei is the corresponding author of this research paper, and the postgraduate Dong De, the senior Ren Kuan and the doctoral student Qiu Xiaolin are equally first authors. Professor Gao Ning's lab, professor Wang Jiawei, doctor Fan Shilong all from the School of Life Science, Tsinghua University participated in parts of the research work. Shanghai Synchrotron Radiation Facility provided timely and effective support for the data collection of crystals. This project is sponsored by National Natural Science Foundation of China, Young Scientist Workshop of Harbin Institute of Technology and other foundations. It is worth mentioning that this is the first time that our school’s undergraduate co-published research paper in this top journal, and this is another paper published in Nature by Huang Zhiwei’s group in the pathogen-host interactions field.
Article link：http://www.nature.com/nature/journal/vaop/ncurrent/full/nature17944.html
Professor Huang and the research group in lab
