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Written | Edited by Xu Ying | Enzymei CRISPR-Cas system is an acquired immune defense system of prokaryotes: After the invader enters the host bacteria, its genes are processed into spacers and stored between the CRISPR repeats to form immunity Memory; when it invades again, the effector complex of prokaryotes responds quickly, recognizing the foreign sequence and inactivating it (Figure 1)
.
The CRISPR-Cas effector complex is composed of Cas protein (CRISPR-associated protein), which can bind to crRNA
.
After the exogenous nucleic acid fragment is integrated into the genome, it can be transcribed under the control of the CRISPR site promoter to synthesize an RNA molecule containing a series of repetitive sequences and spacer sequences, namely the precursor CRISPR RNA (pre-crRNA)
.
Subsequently, the cas protein in the bacteria further processes it and cuts it at a specific site of the CRISPR repetitive sequence to form a CRISPR RNA consisting of a repetitive sequence and a spacer sequence, called crRNA for short
.
The crRNA and the foreign target nucleic acid can complementarily bind, and when the traces of the intruder are detected, the immune response is initiated
.
Figure 1.
CRISPR-Cas adaptive immunity
.
(A) Adaptation: Cas1-Cas2 complex obtains exogenous fragments and integrates them into the CRISPR gene cluster; (B) Expression: CRISPR gene cluster is translated and processed to form crRNA, and its related Cas protein, namely Cas effector nucleic acid Enzymes are expressed, and the latter two are processed and assembled to form a ribonucleic acid complex—monitoring complex; (C) Interference: Effector nuclease complements the exogenous nucleic acid through crRNA and degrades the exogenous fragment [1]
.
Type III CRISPR-Cas immunity is widespread in prokaryotes and is usually mediated by multi-subunit effector complexes
.
Recently, a new CRISPR-Cas subtype similar to the type III system was predicted by bioinformatics and classified as type III-E
.
The effector protein of a typical type III system is composed of multiple protein subunits, and the effector protein of type III-E is formed by the fusion of multiple repeat associated mysterious protein (RAMP) domains, so it is also called It is gRAMP (giant RAMP)
.
gRAMP has 1300-1900 amino acids and is the largest single-subunit effector protein discovered so far
.
TPR-CHAT (tetratrico-peptide repeat, TPR) is predicted to be a protease from the caspase family.
Its members usually catalyze the hydrolysis of specific peptide bonds in the target protein.
It is a caspase-like peptidase whose function remains to be identified
.
On August 27, 2021, the team of Stan JJ Brouns at Delft University of Technology in the Netherlands published an article The gRAMP CRISPR-Cas effector is an RNA endonuclease complexed with a caspase-like peptidase in Science, revealing from The type III-E effector protein (Sb-gRAMP) of anammox bacteria (Candidatus "Scalindua brodae") is a kind of endoribonuclease that can form a complex with TPR-CHAT, which may be activated by target RNA The protease activity of TPR-CHAT peptidase obtains virus immunity
.
The researchers found that the spacer sequence inserted into the type III-E CRISPR gene cluster preferentially targets the coding strand of the open reading frame, indicating that gRAMP activity may interact with the mRNA of the invader
.
Some type III-E loci carry Cas1 fused to reverse transcriptase, suggesting that the mechanism of type III-E acquisition may be the acquisition of spacer sequences from RNA
.
But the strange thing is that the gRAMP gene cluster lacks the auxiliary nuclease gene, but it often co-occurs with the gene encoding the TPR-CHAT protein
.
Therefore, the researchers speculated that there is a certain functional relationship between the caspase family and the antiviral activity of the CRISPR-Cas system III-E type, and verified it in this study
.
The researchers co-expressed Sb-gRAMP and crRNA (containing 5 spacer sequences) using the E.
coli system, and obtained high-purity Sb-gRAMP by Strep affinity chromatography, heparin affinity chromatography, and gel retardation chromatography (SEC).
gRAMP-crRNA complex
.
Multi-angle light scattering (MALS) analysis showed that the resulting complex was formed by combining an Sb-gRAMP monomer with 44-60 nt ssRNA
.
The "Scalindua brodae" type III-E locus contains a CRISPR gene cluster, which consists of 36 nt repeats and 11 spacers
.
In order to further analyze the characteristics of mature crRNA, the researchers used PCR amplification, RNA extraction and RNAseq experiments to prove that the RNA contains a 27-28nt long 5'-handle with a 14nt conserved repeat sequence at the end.
Most mature crRNAs are Cutting
.
Researchers found that Sb-gRAMP can bind to crRNAs with different copy spacing sequences and lacks genes involved in pre-crRNA processing.
Therefore, it is speculated that Sb-gRAMP may be similar to Cas12 and Cas13 and can process pre-crRNA on its own
.
Sb-gRAMP can recognize target RNA and cut single-stranded RNA at two specific sites 6nt apart, and has no cleavage activity on ssDNA or non-complementary ssRNA
.
Therefore, researchers believe that Sb-gRAMP is a sequence and position-specific crRNA-guided endoribonuclease whose RNase activity depends on metal ions
.
Because the tetrapeptide repeat (TPR) domain is usually involved in protein-protein interactions and the formation of protein complexes
.
Therefore, the researchers co-expressed TPR-CHAT and Sb-gRAMP-crRNA with different fusion tags in the cell, and used each other as a bait.
Combined with the results of the MALS experiment, they found that Sb-gRAMP-crRNA can be 1:1 with TPR-CHAT.
The ratio of CRISPR-guided Caspase is combined to form a stable complex, and this complex complex is named Craspase (CRISPR-guided Caspase)
.
The type III-E gRAMP effector protein has both the characteristics of the type I CRISPR-Cas system (multiple domains, 6 nt cleavage distance, and independent PFS and RNA cleavage sites), and the characteristics of the type II CRISPR-Cas system (effector protein Is a single protein), which obscures the traditional classification of the CRISPR-Cas system
.
Although gRAMP-crRNA has the ability to specifically cleave RNA and can contribute to antiviral defense, the main role of target RNA recognition and cleavage in the III-E CRISPR-Cas system is to act as a switch for Caspase
.
In other type III CRISPR-Cas systems, RNA targeting can regulate the production of second messengers, thereby regulating the activity of auxiliary nucleases
.
However, the study found that Sb-gRAMP-crRNA targets RNA and forms a stable complex with TPR-CHAT to create a model, in which Sb-gRAMP-crRNA does not use a second messenger, but is allosteric when the target RNA is recognized Induces peptidase activity, thereby triggering a specific immune response
.
Original link: https://science.
sciencemag.
org/content/early/2021/08/25/science.
abk2718 Platemaker: Eleven References 1.
Gavin J.
Knott, Jennifer A.
Doudna.
CRISPR-Cas guides the future of genetic engineering.
DOI: 10.
1126/science.
aat5011 Reprint Instructions [Original Articles] BioArt original articles are welcome to be shared by individuals.
Reprinting is prohibited without permission.
The copyrights of all published works are owned by BioArt
.
BioArt reserves all statutory rights and offenders must be investigated
.
.
The CRISPR-Cas effector complex is composed of Cas protein (CRISPR-associated protein), which can bind to crRNA
.
After the exogenous nucleic acid fragment is integrated into the genome, it can be transcribed under the control of the CRISPR site promoter to synthesize an RNA molecule containing a series of repetitive sequences and spacer sequences, namely the precursor CRISPR RNA (pre-crRNA)
.
Subsequently, the cas protein in the bacteria further processes it and cuts it at a specific site of the CRISPR repetitive sequence to form a CRISPR RNA consisting of a repetitive sequence and a spacer sequence, called crRNA for short
.
The crRNA and the foreign target nucleic acid can complementarily bind, and when the traces of the intruder are detected, the immune response is initiated
.
Figure 1.
CRISPR-Cas adaptive immunity
.
(A) Adaptation: Cas1-Cas2 complex obtains exogenous fragments and integrates them into the CRISPR gene cluster; (B) Expression: CRISPR gene cluster is translated and processed to form crRNA, and its related Cas protein, namely Cas effector nucleic acid Enzymes are expressed, and the latter two are processed and assembled to form a ribonucleic acid complex—monitoring complex; (C) Interference: Effector nuclease complements the exogenous nucleic acid through crRNA and degrades the exogenous fragment [1]
.
Type III CRISPR-Cas immunity is widespread in prokaryotes and is usually mediated by multi-subunit effector complexes
.
Recently, a new CRISPR-Cas subtype similar to the type III system was predicted by bioinformatics and classified as type III-E
.
The effector protein of a typical type III system is composed of multiple protein subunits, and the effector protein of type III-E is formed by the fusion of multiple repeat associated mysterious protein (RAMP) domains, so it is also called It is gRAMP (giant RAMP)
.
gRAMP has 1300-1900 amino acids and is the largest single-subunit effector protein discovered so far
.
TPR-CHAT (tetratrico-peptide repeat, TPR) is predicted to be a protease from the caspase family.
Its members usually catalyze the hydrolysis of specific peptide bonds in the target protein.
It is a caspase-like peptidase whose function remains to be identified
.
On August 27, 2021, the team of Stan JJ Brouns at Delft University of Technology in the Netherlands published an article The gRAMP CRISPR-Cas effector is an RNA endonuclease complexed with a caspase-like peptidase in Science, revealing from The type III-E effector protein (Sb-gRAMP) of anammox bacteria (Candidatus "Scalindua brodae") is a kind of endoribonuclease that can form a complex with TPR-CHAT, which may be activated by target RNA The protease activity of TPR-CHAT peptidase obtains virus immunity
.
The researchers found that the spacer sequence inserted into the type III-E CRISPR gene cluster preferentially targets the coding strand of the open reading frame, indicating that gRAMP activity may interact with the mRNA of the invader
.
Some type III-E loci carry Cas1 fused to reverse transcriptase, suggesting that the mechanism of type III-E acquisition may be the acquisition of spacer sequences from RNA
.
But the strange thing is that the gRAMP gene cluster lacks the auxiliary nuclease gene, but it often co-occurs with the gene encoding the TPR-CHAT protein
.
Therefore, the researchers speculated that there is a certain functional relationship between the caspase family and the antiviral activity of the CRISPR-Cas system III-E type, and verified it in this study
.
The researchers co-expressed Sb-gRAMP and crRNA (containing 5 spacer sequences) using the E.
coli system, and obtained high-purity Sb-gRAMP by Strep affinity chromatography, heparin affinity chromatography, and gel retardation chromatography (SEC).
gRAMP-crRNA complex
.
Multi-angle light scattering (MALS) analysis showed that the resulting complex was formed by combining an Sb-gRAMP monomer with 44-60 nt ssRNA
.
The "Scalindua brodae" type III-E locus contains a CRISPR gene cluster, which consists of 36 nt repeats and 11 spacers
.
In order to further analyze the characteristics of mature crRNA, the researchers used PCR amplification, RNA extraction and RNAseq experiments to prove that the RNA contains a 27-28nt long 5'-handle with a 14nt conserved repeat sequence at the end.
Most mature crRNAs are Cutting
.
Researchers found that Sb-gRAMP can bind to crRNAs with different copy spacing sequences and lacks genes involved in pre-crRNA processing.
Therefore, it is speculated that Sb-gRAMP may be similar to Cas12 and Cas13 and can process pre-crRNA on its own
.
Sb-gRAMP can recognize target RNA and cut single-stranded RNA at two specific sites 6nt apart, and has no cleavage activity on ssDNA or non-complementary ssRNA
.
Therefore, researchers believe that Sb-gRAMP is a sequence and position-specific crRNA-guided endoribonuclease whose RNase activity depends on metal ions
.
Because the tetrapeptide repeat (TPR) domain is usually involved in protein-protein interactions and the formation of protein complexes
.
Therefore, the researchers co-expressed TPR-CHAT and Sb-gRAMP-crRNA with different fusion tags in the cell, and used each other as a bait.
Combined with the results of the MALS experiment, they found that Sb-gRAMP-crRNA can be 1:1 with TPR-CHAT.
The ratio of CRISPR-guided Caspase is combined to form a stable complex, and this complex complex is named Craspase (CRISPR-guided Caspase)
.
The type III-E gRAMP effector protein has both the characteristics of the type I CRISPR-Cas system (multiple domains, 6 nt cleavage distance, and independent PFS and RNA cleavage sites), and the characteristics of the type II CRISPR-Cas system (effector protein Is a single protein), which obscures the traditional classification of the CRISPR-Cas system
.
Although gRAMP-crRNA has the ability to specifically cleave RNA and can contribute to antiviral defense, the main role of target RNA recognition and cleavage in the III-E CRISPR-Cas system is to act as a switch for Caspase
.
In other type III CRISPR-Cas systems, RNA targeting can regulate the production of second messengers, thereby regulating the activity of auxiliary nucleases
.
However, the study found that Sb-gRAMP-crRNA targets RNA and forms a stable complex with TPR-CHAT to create a model, in which Sb-gRAMP-crRNA does not use a second messenger, but is allosteric when the target RNA is recognized Induces peptidase activity, thereby triggering a specific immune response
.
Original link: https://science.
sciencemag.
org/content/early/2021/08/25/science.
abk2718 Platemaker: Eleven References 1.
Gavin J.
Knott, Jennifer A.
Doudna.
CRISPR-Cas guides the future of genetic engineering.
DOI: 10.
1126/science.
aat5011 Reprint Instructions [Original Articles] BioArt original articles are welcome to be shared by individuals.
Reprinting is prohibited without permission.
The copyrights of all published works are owned by BioArt
.
BioArt reserves all statutory rights and offenders must be investigated
.
