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The Type III-E CRISPR-Cas system uses a single multidomain 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 Type III-E CRISPR-Cas system are unknown
.
On October 27, 2022, in a new study published in Nature Microbiology, Zhang Heng's team from Tianjin Medical University and the Wuhan Institute of Virology, Chinese Academy of Sciences By analyzing the cryo-EM structure of Cas7-11 complexes in different states, Deng Zengqin's team used a large number of biochemical experiments to elucidate the mechanism of Cas7-11 processing precursor CRISPR RNA (pre-crRNA) and identifying and cleavaging target RNA.
The study also found that Cas7-11 can cause conformational changes in Csx29 after recognizing target RNA, which is likely to activate its protease activity to exert immune function
.
The CRISPR-Cas system, an adaptive immune system in bacteria and archaea resistant to invasion by mobile genetic primitives, can be divided into two categories, Class 1: protein complexes consisting of multiple effector proteins perform functions, including I, Types III and IV; Class 2 is functioned by individual effector proteins, including types
II, V and VI.
Recently, researchers identified a novel III-E subtype, the CRISPR-Cas system
.
This system has high activity and low toxicity
when RNA is targeted by mammalian cells.
Unlike the previously discovered polysubunit type III system, it consists of the fusion of 4 Cas7 and 1 Cas11 domains to form a large effector protein Cas7-11/gRAMP, which cleaves
the target RNA under the guidance of crRNA 。 Cas7-11 can interact with a Caspase-like protease (Csx29/TPR-CHAT) (ref 3) to form the 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 used cryo-EM to resolve the Cas7-11-crRNA (SbCas7-11-crRNA) binary complex structure of the Candidatus 'Scalindua brodae' strain.
Cas7-11 was found to consist of four structurally similar Cas7s as well as a Cas11 and an IPD domain (Figure 1).
IPD is an insertion sequence located in the middle of Cas7.
4, and its structure and function are currently unclear
.
However, after the authors deleted IPD, it did not affect the recognition and cleavage of target RNA, suggesting that miniaturized gene editing tools
could be built by deleting IPD.
Figure 1 Gene structure
of type III-E CRISPR-Cas system In the traditional type III CRISPR-Cas system, Cas6 protein is responsible for pre-crRNA processing and maturation.
The Cas7-11 protein itself has the corresponding pre-crRNA processing ability
.
The Cas7-11–crRNA structure shows that the repeats of mature crRNAs bind to the Cas7.
1 domain, so it is speculated that the Cas7.
1 domain may have the ability to
process pre-crRNAs.
Next, the authors designed a series of mutation experiments to find the active site
of Cas7.
1.
Due to the instability of monomeric SbCas7-11, the authors selected DiCas7-11, a homologous protein from Desulfonema ishimotonii species, for experiments
.
The results showed that the 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 Cas7-11 congeners, but H43 is replaced by threonine residue (T45) in SbCas7-11, and Y55 is replaced
by phenylalanine residue (F57).
Figure 2 In vitro pre-crRNA cleavage assay
for wild-type (WT) and mutant DiCas7-11 proteins.
In parentheses, SbCas7-11 corresponds to the residue (green) Cas7-11 has two target RNA cleavage sites separated 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 structure of the Cas7.
2 and Cas7.
3 domains, respectively, suggesting that target RNA cleavage is likely to occur near
these two sites.
In III-A/B systems, the Csm3/Cmr4 subunit exerts RNase activity
through a conserved aspartate residue in the catalytic loop.
By comparing the structure of 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 eliminates 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, the mutation of the acidic residue D547 in the same catalytic pocket virtually eliminated the cleavage activity of SbCas7-11 at site 1 (Figure 3).
More interestingly, the sequence alignment showed that in most Cas7-11 congeners, only one acidic amino acid
generally appears at these two corresponding positions.
For example, SbCas7-11 has S457-D547, while DiCas7-11 is D429-N518
in the corresponding position.
Therefore, the authors proposed the hypothesis that amino acids in these two locations may have functional redundancy, and made loss of function and gain-of-function mutations to test this hypothesis
.
As expected, the results suggest that these two active site acid residues of 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
for Cas7.
3 domain mutation.
In vitro target RNA cleavage
for Cas7.
2 domain mutations.
Right, Sequence alignment
of Cas7-11 homologs In order to study the mechanism by which Cas7-11 regulates Csx29, the authors analyzed the Cas7-11-crRNA-Csx29 ternary complex and Cas7-11-crRNA-target EM structure
of RNA-Csx29 quaternary complex.
The structure reveals that Csx29 undergoes a significant conformational change in the presence of target RNA: the TPR region is closer to Cas7-11, while the protease PS domain is far from Cas7-11, making the two catalytic residues of Csx29, H585 and C627, spatially closer together, exposing their catalytic pockets (Figure 4).
Therefore, it is likely that the binding of target RNA can stimulate Csx29 protease activity to synergize with Cas7-11 against the invasion
of foreign nucleic acids.
The team's follow-up work on the determination of Csx29 protease activity and the identification of cleavage substrates also confirmed this inference (this part of the research work is under revision).
Fig.
4 Conformational changes of Csx29 when unbound target RNA and target RNA were conjugated, and the mechanism of Cas7-11 processing maturation pre-crRNA and cleavage target RNA was elucidated and the regulatory mechanism of Cas7-11 on Csx29 activity (Figure 5).
。 This research has greatly contributed to the understanding of the CRISPR system and provided a structural basis
for the engineering 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
paper link:
for RNA-targeted applications.
The structure and molecular mechanisms of the Type III-E CRISPR-Cas system are unknown
.
On October 27, 2022, in a new study published in Nature Microbiology, Zhang Heng's team from Tianjin Medical University and the Wuhan Institute of Virology, Chinese Academy of Sciences By analyzing the cryo-EM structure of Cas7-11 complexes in different states, Deng Zengqin's team used a large number of biochemical experiments to elucidate the mechanism of Cas7-11 processing precursor CRISPR RNA (pre-crRNA) and identifying and cleavaging target RNA.
The study also found that Cas7-11 can cause conformational changes in Csx29 after recognizing target RNA, which is likely to activate its protease activity to exert immune function
.
The CRISPR-Cas system, an adaptive immune system in bacteria and archaea resistant to invasion by mobile genetic primitives, can be divided into two categories, Class 1: protein complexes consisting of multiple effector proteins perform functions, including I, Types III and IV; Class 2 is functioned by individual effector proteins, including types
II, V and VI.
Recently, researchers identified a novel III-E subtype, the CRISPR-Cas system
.
This system has high activity and low toxicity
when RNA is targeted by mammalian cells.
Unlike the previously discovered polysubunit type III system, it consists of the fusion of 4 Cas7 and 1 Cas11 domains to form a large effector protein Cas7-11/gRAMP, which cleaves
the target RNA under the guidance of crRNA 。 Cas7-11 can interact with a Caspase-like protease (Csx29/TPR-CHAT) (ref 3) to form the 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 used cryo-EM to resolve the Cas7-11-crRNA (SbCas7-11-crRNA) binary complex structure of the Candidatus 'Scalindua brodae' strain.
Cas7-11 was found to consist of four structurally similar Cas7s as well as a Cas11 and an IPD domain (Figure 1).
IPD is an insertion sequence located in the middle of Cas7.
4, and its structure and function are currently unclear
.
However, after the authors deleted IPD, it did not affect the recognition and cleavage of target RNA, suggesting that miniaturized gene editing tools
could be built by deleting IPD.
Figure 1 Gene structure
of type III-E CRISPR-Cas system In the traditional type III CRISPR-Cas system, Cas6 protein is responsible for pre-crRNA processing and maturation.
The Cas7-11 protein itself has the corresponding pre-crRNA processing ability
.
The Cas7-11–crRNA structure shows that the repeats of mature crRNAs bind to the Cas7.
1 domain, so it is speculated that the Cas7.
1 domain may have the ability to
process pre-crRNAs.
Next, the authors designed a series of mutation experiments to find the active site
of Cas7.
1.
Due to the instability of monomeric SbCas7-11, the authors selected DiCas7-11, a homologous protein from Desulfonema ishimotonii species, for experiments
.
The results showed that the 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 Cas7-11 congeners, but H43 is replaced by threonine residue (T45) in SbCas7-11, and Y55 is replaced
by phenylalanine residue (F57).
Figure 2 In vitro pre-crRNA cleavage assay
for wild-type (WT) and mutant DiCas7-11 proteins.
In parentheses, SbCas7-11 corresponds to the residue (green) Cas7-11 has two target RNA cleavage sites separated 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 structure of the Cas7.
2 and Cas7.
3 domains, respectively, suggesting that target RNA cleavage is likely to occur near
these two sites.
In III-A/B systems, the Csm3/Cmr4 subunit exerts RNase activity
through a conserved aspartate residue in the catalytic loop.
By comparing the structure of 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 eliminates 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, the mutation of the acidic residue D547 in the same catalytic pocket virtually eliminated the cleavage activity of SbCas7-11 at site 1 (Figure 3).
More interestingly, the sequence alignment showed that in most Cas7-11 congeners, only one acidic amino acid
generally appears at these two corresponding positions.
For example, SbCas7-11 has S457-D547, while DiCas7-11 is D429-N518
in the corresponding position.
Therefore, the authors proposed the hypothesis that amino acids in these two locations may have functional redundancy, and made loss of function and gain-of-function mutations to test this hypothesis
.
As expected, the results suggest that these two active site acid residues of 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
for Cas7.
3 domain mutation.
In vitro target RNA cleavage
for Cas7.
2 domain mutations.
Right, Sequence alignment
of Cas7-11 homologs In order to study the mechanism by which Cas7-11 regulates Csx29, the authors analyzed the Cas7-11-crRNA-Csx29 ternary complex and Cas7-11-crRNA-target EM structure
of RNA-Csx29 quaternary complex.
The structure reveals that Csx29 undergoes a significant conformational change in the presence of target RNA: the TPR region is closer to Cas7-11, while the protease PS domain is far from Cas7-11, making the two catalytic residues of Csx29, H585 and C627, spatially closer together, exposing their catalytic pockets (Figure 4).
Therefore, it is likely that the binding of target RNA can stimulate Csx29 protease activity to synergize with Cas7-11 against the invasion
of foreign nucleic acids.
The team's follow-up work on the determination of Csx29 protease activity and the identification of cleavage substrates also confirmed this inference (this part of the research work is under revision).
Fig.
4 Conformational changes of Csx29 when unbound target RNA and target RNA were conjugated, and the mechanism of Cas7-11 processing maturation pre-crRNA and cleavage target RNA was elucidated and the regulatory mechanism of Cas7-11 on Csx29 activity (Figure 5).
。 This research has greatly contributed to the understanding of the CRISPR system and provided a structural basis
for the engineering 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
paper link: