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The III-E type CRISPR-Cas system uses a single multi-domain effector called Cas7-11 (also known as gRAMP) to cleave RNA and bind to the caspase-like protease Csx29, showing good potential
for RNA-targeted applications.
The structure and molecular mechanisms of the III-E CRISPR-Cas system are unknown
.
On October 27, 2022, the team of Zhang Heng of Tianjin Medical University and the team of Deng Zengqin of the Wuhan Institute of Virology, Chinese Academy of Sciences published a paper entitled "Structure and function of a bacterial type III-E CRISPR–Cas7-11 complex" in the journal Nature Microbiology The research paper, which uses a large number of biochemical experiments to elucidate the mechanism
by which Cas7-11 processes the precursor CRISPR RNA (pre-crRNA) to recognize and cut target RNA by resolving the cryo-EM structure of the Cas7-11 complex in different states.
The study also found that Cas7-11, after recognizing target RNA, can cause conformational changes in Csx29, which is likely to activate its protease activity to exert immune function
.
II, V and VI.
Recently researchers identified a novel III-E subtype, the CRISPR-Cas system (ref 1).
The system has high activity and low toxicity (ref 2)
when targeting RNA in mammalian cells.
Unlike the previously discovered multi-subunit type III system, it consists of four Cas7 and one Cas11 domain fused to form a large effector protein, Cas7-11/gRAMP, which cleaves
the target RNA under the guidance of crRNA 。 Cas7-11 interacts with a Caspase-like protease (Csx29/TPR-CHAT) (ref 3) to form a CRISPR-guided Caspase complex (Craspase), so the system may have both nuclease and protease activity, suggesting a novel mechanism
against phage infection.
In this latest study, the authors first analyzed the binary complex structure of the Cas7-11-crRNA (SbCas7-11-crRNA) strain of the Candidatus 'Scalindua brodae' strain with the help of cryo-EM and found that Cas7-11 consists of four structurally similar Cas7s as well as a Cas11 and an IPD domain (Figure 1).
。 IPD is an insertion sequence in the middle of Cas7.
4, and its structure and function are currently unknown
.
However, after the authors deleted the IPD, it did not affect the recognition and cleavage of the target RNA, suggesting that miniaturized gene-editing tools
could be constructed by deleting the IPD.
Fig.
1 Gene structure of III-E CRISPR-Cas system In the traditional type III CRISPR-Cas system, Cas6 protein is responsible for pre-crRNA processing maturation, and Cas7-11 protein itself has the corresponding pre-crRNA processing capacity
。 The Cas7-11–crRNA structure shows that the repetitive sequence of the mature crRNA binds to the Cas7.
1 domain, so it is speculated that the Cas7.
1 domain may have the ability to
process pre-crRNA.
Next, the authors designed a series of mutation experiments to find the active site
of Cas7.
1.
Due to the instability of the monomeric SbCas7-11, the authors selected DiCas7-11, a homologous protein from the Desulfonema ishimotonii species
.
The results showed that all four mutants of DiCas7-11 (W20A, R26A, H43A and Y55A) could significantly hinder the processing of pre-crRNA (Figure 2), demonstrating the important role
of the Cas7.
1 domain in pre-crRNA cleavage.
Among them, W20 and R26 are strictly conserved in the Cas7-11 homologous, but H43 is replaced by threonine residue (T45) and Y55 by phenylalanine residue (F57) in SbCas7-11
.
Fig.
2 In vitro pre-crRNA cleavage experiment
of wild-type (WT) and mutant DiCas7-11 proteins.
In parentheses, SbCas7-11 corresponds to residue (green) Cas7-11 has two target RNA cleavage sites, spaced by 6 nucleotides, called site 1 and site 2
.
The Cas7-11-crRNA-target RNA ternary complex structure found that two nucleotides in the crRNA sequence, 4A and 10U, were flipped by the β-finger structures of the Cas7.
2 and Cas7.
3 domains, respectively, suggesting that target RNA cleavage is likely to occur near
these two sites.
In the III-A/B system, the Csm3/Cmr4 subunit exerts RNase activity
by catalyzing a conserved aspartate residue in the loop.
By comparing the structure of the SbCas7-11 and Csm complexes, the authors found that there is a corresponding conserved aspartate residue (D698)
in Cas7.
3.
As expected, the D698A mutation virtually eliminated target RNA cleavage at 2 sites (Figure 3 left).
However, the corresponding position in Cas7.
2 contains a non-conserved serine residue (S457), and the S457A mutation has little effect on cleavage at site 1
.
Interestingly, mutations in acidic residue D547 in the same catalytic pocket virtually eliminate the cleavage activity of SbCas7-11 at site 1 (Figure 3).
More interestingly, sequence alignment shows that in most Cas7-11 congeners, only one acidic amino acid
generally appears in these two corresponding positions.
For example, SbCas7-11 has S457-D547, while DiCas7-11 has D429-N518
in the corresponding position.
Therefore, the authors put forward the hypothesis that amino acids at these two locations may have functional redundancy, and made loss of function and function acquisition mutations to test this hypothesis
.
As expected, the results show that these two active site acidic residues in the Cas7.
2 catalytic pocket may be functionally equivalent: either aspartate residue at either location can cleave the target RNA (Figure 3 right).
Figure 3 Left, in vitro target RNA cleavage
of Cas7.
3 domain mutations.
In vitro, in vitro target RNA cleavage
of Cas7.
2 domain mutations.
Right, sequence alignment of Cas7-11 homologs To investigate the mechanism of how Cas7-11 regulates Csx29, the authors resolved the electron microscopy structure
of the Cas7-11-crRNA-Csx29 ternary complex and the Cas7-11-crRNA-target RNA-Csx29 quaternary complex 。 The structure revealed that Csx29 underwent significant conformational changes in the presence of target RNA: the TPR region was close to Cas7-11, while the protease PS domain was far away from Cas7-11, making the two catalytic residues of Csx29, H585 and C627, spatially closer together, exposing its catalytic pocket (Figure 4).
Therefore, it is likely that the binding of target RNA can stimulate Csx29 protease activity, thereby countering foreign nucleic acid invasion
in synergy with Cas7-11.
This inference was confirmed by the team's follow-up work on the determination of Csx29 protease activity and the identification of the cleavage substrate (part of the work is under revision).
Fig.
4 Conformational changes when Csx29 does not bind to target RNA and binds target RNA This study describes the mechanism of Cas7-11 processing mature pre-crRNA and cleavage of target RNA, as well as the regulatory mechanism of Cas7-11 on Csx29 activity (Fig.
5).
This study has greatly advanced the understanding of the CRISPR system and provided a structural basis
for the engineered modification of Cas7-11 as a safe and efficient targeted RNA editing tool.
At the same time, the RNA-guided protease activity of this system may bring new perspectives and new tools
to life science research.
Figure 5 Article pattern diagram
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