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eicsm6  (Addgene inc)


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    Addgene inc eicsm6
    Eicsm6, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/eicsm6/pC0073+EiCsm6+His6-TwinStrep-SUMO-BsaI+(Plasmid+%23115268)/pm34354262-381-9-20
    Average 92 stars, based on 2 article reviews
    eicsm6 - by Bioz Stars, 2026-09
    92/100 stars

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    Related Articles

    Sequencing:

    Article Title: Activation and self-inactivation mechanisms of the cyclic oligoadenylate-dependent CRISPR ribonuclease Csm6
    Article Snippet: .. The gene sequence of EiCsm6 was amplified from genomic DNA ( Enterococcus italicus DSM-15952) by PCR and cloned into a 1B vector (Addgene 29653) using ligation-independent cloning resulting in constructs carrying an N-terminal hexahistidine tag followed by a Tobacco Etch Virus (TEV) protease cleavage site. ..

    Article Title: Type III CRISPR-Cas systems generate cyclic oligoadenylate second messengers to activate Csm6 RNases
    Article Snippet: .. DNA sequences encoding for TtCsm6 ( Thermus thermophilus DSM-579), EiCsm6 ( Enterococcus italicus DSM-15952) and MtCsm6 ( Methanothermobacter thermoautotrophicus DSM-1053) were amplified from their respective genomic DNAs by a standard PCR protocol and were subsequently cloned into a 2HR-T vector (Addgene #29718) using ligation-independent cloning, resulting in constructs carrying an N-terminal hexahistidine tag followed by a StrepII tag, a Tobacco Etch Virus (TEV) protease cleavage site, and the Csm6 polypeptide sequence. .. To obtain a plasmid vector for heterologous expression a functional EiCsm(1-5) interference complex from the E. italicus DSM-15952 type III-A CRISPR locus, a DNA fragment spanning from the cas10 gene to the end of the second spacer (GenBank GL622241.1 ; complement 1999065-1991480) was amplified from E. italicus genomic DNA using primers oCGD208 and oCGD209 and cloned into a p7XNH3 vector using FX cloning , resulting in the pCGD253 plasmid.

    Article Title: Type III CRISPR-Cas systems produce cyclic oligoadenylate second messengers.
    Article Snippet: .. DNA sequences encoding for TtCsm6 (Thermus thermophilus DSM-579), EiCsm6 (Enterococcus italicus DSM-15952) and MtCsm6 (Methanothermobacter thermoautotrophicus DSM-1053) were amplified from their respective genomic DNAs by a standard PCR protocol and were subsequently cloned into a 2HR-T vector (Addgene #29718) using ligation-independent cloning, resulting in constructs carrying an N-terminal hexa-histidine tag followed by a StrepII tag, a Tobacco Etch Virus (TEV) protease cleavage site, and the Csm6 polypeptide sequence. ..

    Article Title: Activation and self-inactivation mechanisms of the cyclic oligoadenylate-dependent CRISPR ribonuclease Csm6
    Article Snippet: .. The gene sequence of EiCsm6 was amplified from genomic DNA ( Enterococcus italicus DSM-15952) by PCR and cloned into a 1B vector (Addgene 29653) using ligation-independent cloning resulting in constructs carrying an N-terminal hexahistidine tag followed by a tobacco etch virus (TEV) protease cleavage site. ..

    Amplification:

    Article Title: Activation and self-inactivation mechanisms of the cyclic oligoadenylate-dependent CRISPR ribonuclease Csm6
    Article Snippet: .. The gene sequence of EiCsm6 was amplified from genomic DNA ( Enterococcus italicus DSM-15952) by PCR and cloned into a 1B vector (Addgene 29653) using ligation-independent cloning resulting in constructs carrying an N-terminal hexahistidine tag followed by a Tobacco Etch Virus (TEV) protease cleavage site. ..

    Article Title: Type III CRISPR-Cas systems generate cyclic oligoadenylate second messengers to activate Csm6 RNases
    Article Snippet: .. DNA sequences encoding for TtCsm6 ( Thermus thermophilus DSM-579), EiCsm6 ( Enterococcus italicus DSM-15952) and MtCsm6 ( Methanothermobacter thermoautotrophicus DSM-1053) were amplified from their respective genomic DNAs by a standard PCR protocol and were subsequently cloned into a 2HR-T vector (Addgene #29718) using ligation-independent cloning, resulting in constructs carrying an N-terminal hexahistidine tag followed by a StrepII tag, a Tobacco Etch Virus (TEV) protease cleavage site, and the Csm6 polypeptide sequence. .. To obtain a plasmid vector for heterologous expression a functional EiCsm(1-5) interference complex from the E. italicus DSM-15952 type III-A CRISPR locus, a DNA fragment spanning from the cas10 gene to the end of the second spacer (GenBank GL622241.1 ; complement 1999065-1991480) was amplified from E. italicus genomic DNA using primers oCGD208 and oCGD209 and cloned into a p7XNH3 vector using FX cloning , resulting in the pCGD253 plasmid.

    Article Title: Type III CRISPR-Cas systems produce cyclic oligoadenylate second messengers.
    Article Snippet: .. DNA sequences encoding for TtCsm6 (Thermus thermophilus DSM-579), EiCsm6 (Enterococcus italicus DSM-15952) and MtCsm6 (Methanothermobacter thermoautotrophicus DSM-1053) were amplified from their respective genomic DNAs by a standard PCR protocol and were subsequently cloned into a 2HR-T vector (Addgene #29718) using ligation-independent cloning, resulting in constructs carrying an N-terminal hexa-histidine tag followed by a StrepII tag, a Tobacco Etch Virus (TEV) protease cleavage site, and the Csm6 polypeptide sequence. ..

    Article Title: Activation and self-inactivation mechanisms of the cyclic oligoadenylate-dependent CRISPR ribonuclease Csm6
    Article Snippet: .. The gene sequence of EiCsm6 was amplified from genomic DNA ( Enterococcus italicus DSM-15952) by PCR and cloned into a 1B vector (Addgene 29653) using ligation-independent cloning resulting in constructs carrying an N-terminal hexahistidine tag followed by a tobacco etch virus (TEV) protease cleavage site. ..

    Polymerase Chain Reaction:

    Article Title: Activation and self-inactivation mechanisms of the cyclic oligoadenylate-dependent CRISPR ribonuclease Csm6
    Article Snippet: .. The gene sequence of EiCsm6 was amplified from genomic DNA ( Enterococcus italicus DSM-15952) by PCR and cloned into a 1B vector (Addgene 29653) using ligation-independent cloning resulting in constructs carrying an N-terminal hexahistidine tag followed by a Tobacco Etch Virus (TEV) protease cleavage site. ..

    Article Title: Type III CRISPR-Cas systems generate cyclic oligoadenylate second messengers to activate Csm6 RNases
    Article Snippet: .. DNA sequences encoding for TtCsm6 ( Thermus thermophilus DSM-579), EiCsm6 ( Enterococcus italicus DSM-15952) and MtCsm6 ( Methanothermobacter thermoautotrophicus DSM-1053) were amplified from their respective genomic DNAs by a standard PCR protocol and were subsequently cloned into a 2HR-T vector (Addgene #29718) using ligation-independent cloning, resulting in constructs carrying an N-terminal hexahistidine tag followed by a StrepII tag, a Tobacco Etch Virus (TEV) protease cleavage site, and the Csm6 polypeptide sequence. .. To obtain a plasmid vector for heterologous expression a functional EiCsm(1-5) interference complex from the E. italicus DSM-15952 type III-A CRISPR locus, a DNA fragment spanning from the cas10 gene to the end of the second spacer (GenBank GL622241.1 ; complement 1999065-1991480) was amplified from E. italicus genomic DNA using primers oCGD208 and oCGD209 and cloned into a p7XNH3 vector using FX cloning , resulting in the pCGD253 plasmid.

    Article Title: Type III CRISPR-Cas systems produce cyclic oligoadenylate second messengers.
    Article Snippet: .. DNA sequences encoding for TtCsm6 (Thermus thermophilus DSM-579), EiCsm6 (Enterococcus italicus DSM-15952) and MtCsm6 (Methanothermobacter thermoautotrophicus DSM-1053) were amplified from their respective genomic DNAs by a standard PCR protocol and were subsequently cloned into a 2HR-T vector (Addgene #29718) using ligation-independent cloning, resulting in constructs carrying an N-terminal hexa-histidine tag followed by a StrepII tag, a Tobacco Etch Virus (TEV) protease cleavage site, and the Csm6 polypeptide sequence. ..

    Article Title: Activation and self-inactivation mechanisms of the cyclic oligoadenylate-dependent CRISPR ribonuclease Csm6
    Article Snippet: .. The gene sequence of EiCsm6 was amplified from genomic DNA ( Enterococcus italicus DSM-15952) by PCR and cloned into a 1B vector (Addgene 29653) using ligation-independent cloning resulting in constructs carrying an N-terminal hexahistidine tag followed by a tobacco etch virus (TEV) protease cleavage site. ..

    Clone Assay:

    Article Title: Activation and self-inactivation mechanisms of the cyclic oligoadenylate-dependent CRISPR ribonuclease Csm6
    Article Snippet: .. The gene sequence of EiCsm6 was amplified from genomic DNA ( Enterococcus italicus DSM-15952) by PCR and cloned into a 1B vector (Addgene 29653) using ligation-independent cloning resulting in constructs carrying an N-terminal hexahistidine tag followed by a Tobacco Etch Virus (TEV) protease cleavage site. ..

    Article Title: Type III CRISPR-Cas systems generate cyclic oligoadenylate second messengers to activate Csm6 RNases
    Article Snippet: .. DNA sequences encoding for TtCsm6 ( Thermus thermophilus DSM-579), EiCsm6 ( Enterococcus italicus DSM-15952) and MtCsm6 ( Methanothermobacter thermoautotrophicus DSM-1053) were amplified from their respective genomic DNAs by a standard PCR protocol and were subsequently cloned into a 2HR-T vector (Addgene #29718) using ligation-independent cloning, resulting in constructs carrying an N-terminal hexahistidine tag followed by a StrepII tag, a Tobacco Etch Virus (TEV) protease cleavage site, and the Csm6 polypeptide sequence. .. To obtain a plasmid vector for heterologous expression a functional EiCsm(1-5) interference complex from the E. italicus DSM-15952 type III-A CRISPR locus, a DNA fragment spanning from the cas10 gene to the end of the second spacer (GenBank GL622241.1 ; complement 1999065-1991480) was amplified from E. italicus genomic DNA using primers oCGD208 and oCGD209 and cloned into a p7XNH3 vector using FX cloning , resulting in the pCGD253 plasmid.

    Article Title: Type III CRISPR-Cas systems produce cyclic oligoadenylate second messengers.
    Article Snippet: .. DNA sequences encoding for TtCsm6 (Thermus thermophilus DSM-579), EiCsm6 (Enterococcus italicus DSM-15952) and MtCsm6 (Methanothermobacter thermoautotrophicus DSM-1053) were amplified from their respective genomic DNAs by a standard PCR protocol and were subsequently cloned into a 2HR-T vector (Addgene #29718) using ligation-independent cloning, resulting in constructs carrying an N-terminal hexa-histidine tag followed by a StrepII tag, a Tobacco Etch Virus (TEV) protease cleavage site, and the Csm6 polypeptide sequence. ..

    Article Title: Activation and self-inactivation mechanisms of the cyclic oligoadenylate-dependent CRISPR ribonuclease Csm6
    Article Snippet: .. The gene sequence of EiCsm6 was amplified from genomic DNA ( Enterococcus italicus DSM-15952) by PCR and cloned into a 1B vector (Addgene 29653) using ligation-independent cloning resulting in constructs carrying an N-terminal hexahistidine tag followed by a tobacco etch virus (TEV) protease cleavage site. ..

    Plasmid Preparation:

    Article Title: Activation and self-inactivation mechanisms of the cyclic oligoadenylate-dependent CRISPR ribonuclease Csm6
    Article Snippet: .. The gene sequence of EiCsm6 was amplified from genomic DNA ( Enterococcus italicus DSM-15952) by PCR and cloned into a 1B vector (Addgene 29653) using ligation-independent cloning resulting in constructs carrying an N-terminal hexahistidine tag followed by a Tobacco Etch Virus (TEV) protease cleavage site. ..

    Article Title: Activation and self-inactivation mechanisms of the cyclic oligoadenylate-dependent CRISPR ribonuclease Csm6
    Article Snippet: .. The gene sequence of EiCsm6 was amplified from genomic DNA ( Enterococcus italicus DSM-15952) by PCR and cloned into a 1B vector (Addgene 29653) using ligation-independent cloning resulting in constructs carrying an N-terminal hexahistidine tag followed by a tobacco etch virus (TEV) protease cleavage site. ..

    Ligation:

    Article Title: Activation and self-inactivation mechanisms of the cyclic oligoadenylate-dependent CRISPR ribonuclease Csm6
    Article Snippet: .. The gene sequence of EiCsm6 was amplified from genomic DNA ( Enterococcus italicus DSM-15952) by PCR and cloned into a 1B vector (Addgene 29653) using ligation-independent cloning resulting in constructs carrying an N-terminal hexahistidine tag followed by a Tobacco Etch Virus (TEV) protease cleavage site. ..

    Article Title: Type III CRISPR-Cas systems generate cyclic oligoadenylate second messengers to activate Csm6 RNases
    Article Snippet: .. DNA sequences encoding for TtCsm6 ( Thermus thermophilus DSM-579), EiCsm6 ( Enterococcus italicus DSM-15952) and MtCsm6 ( Methanothermobacter thermoautotrophicus DSM-1053) were amplified from their respective genomic DNAs by a standard PCR protocol and were subsequently cloned into a 2HR-T vector (Addgene #29718) using ligation-independent cloning, resulting in constructs carrying an N-terminal hexahistidine tag followed by a StrepII tag, a Tobacco Etch Virus (TEV) protease cleavage site, and the Csm6 polypeptide sequence. .. To obtain a plasmid vector for heterologous expression a functional EiCsm(1-5) interference complex from the E. italicus DSM-15952 type III-A CRISPR locus, a DNA fragment spanning from the cas10 gene to the end of the second spacer (GenBank GL622241.1 ; complement 1999065-1991480) was amplified from E. italicus genomic DNA using primers oCGD208 and oCGD209 and cloned into a p7XNH3 vector using FX cloning , resulting in the pCGD253 plasmid.

    Article Title: Type III CRISPR-Cas systems produce cyclic oligoadenylate second messengers.
    Article Snippet: .. DNA sequences encoding for TtCsm6 (Thermus thermophilus DSM-579), EiCsm6 (Enterococcus italicus DSM-15952) and MtCsm6 (Methanothermobacter thermoautotrophicus DSM-1053) were amplified from their respective genomic DNAs by a standard PCR protocol and were subsequently cloned into a 2HR-T vector (Addgene #29718) using ligation-independent cloning, resulting in constructs carrying an N-terminal hexa-histidine tag followed by a StrepII tag, a Tobacco Etch Virus (TEV) protease cleavage site, and the Csm6 polypeptide sequence. ..

    Article Title: Activation and self-inactivation mechanisms of the cyclic oligoadenylate-dependent CRISPR ribonuclease Csm6
    Article Snippet: .. The gene sequence of EiCsm6 was amplified from genomic DNA ( Enterococcus italicus DSM-15952) by PCR and cloned into a 1B vector (Addgene 29653) using ligation-independent cloning resulting in constructs carrying an N-terminal hexahistidine tag followed by a tobacco etch virus (TEV) protease cleavage site. ..

    Cloning:

    Article Title: Activation and self-inactivation mechanisms of the cyclic oligoadenylate-dependent CRISPR ribonuclease Csm6
    Article Snippet: .. The gene sequence of EiCsm6 was amplified from genomic DNA ( Enterococcus italicus DSM-15952) by PCR and cloned into a 1B vector (Addgene 29653) using ligation-independent cloning resulting in constructs carrying an N-terminal hexahistidine tag followed by a Tobacco Etch Virus (TEV) protease cleavage site. ..

    Article Title: Type III CRISPR-Cas systems generate cyclic oligoadenylate second messengers to activate Csm6 RNases
    Article Snippet: .. DNA sequences encoding for TtCsm6 ( Thermus thermophilus DSM-579), EiCsm6 ( Enterococcus italicus DSM-15952) and MtCsm6 ( Methanothermobacter thermoautotrophicus DSM-1053) were amplified from their respective genomic DNAs by a standard PCR protocol and were subsequently cloned into a 2HR-T vector (Addgene #29718) using ligation-independent cloning, resulting in constructs carrying an N-terminal hexahistidine tag followed by a StrepII tag, a Tobacco Etch Virus (TEV) protease cleavage site, and the Csm6 polypeptide sequence. .. To obtain a plasmid vector for heterologous expression a functional EiCsm(1-5) interference complex from the E. italicus DSM-15952 type III-A CRISPR locus, a DNA fragment spanning from the cas10 gene to the end of the second spacer (GenBank GL622241.1 ; complement 1999065-1991480) was amplified from E. italicus genomic DNA using primers oCGD208 and oCGD209 and cloned into a p7XNH3 vector using FX cloning , resulting in the pCGD253 plasmid.

    Article Title: Type III CRISPR-Cas systems produce cyclic oligoadenylate second messengers.
    Article Snippet: .. DNA sequences encoding for TtCsm6 (Thermus thermophilus DSM-579), EiCsm6 (Enterococcus italicus DSM-15952) and MtCsm6 (Methanothermobacter thermoautotrophicus DSM-1053) were amplified from their respective genomic DNAs by a standard PCR protocol and were subsequently cloned into a 2HR-T vector (Addgene #29718) using ligation-independent cloning, resulting in constructs carrying an N-terminal hexa-histidine tag followed by a StrepII tag, a Tobacco Etch Virus (TEV) protease cleavage site, and the Csm6 polypeptide sequence. ..

    Article Title: Activation and self-inactivation mechanisms of the cyclic oligoadenylate-dependent CRISPR ribonuclease Csm6
    Article Snippet: .. The gene sequence of EiCsm6 was amplified from genomic DNA ( Enterococcus italicus DSM-15952) by PCR and cloned into a 1B vector (Addgene 29653) using ligation-independent cloning resulting in constructs carrying an N-terminal hexahistidine tag followed by a tobacco etch virus (TEV) protease cleavage site. ..

    Construct:

    Article Title: Activation and self-inactivation mechanisms of the cyclic oligoadenylate-dependent CRISPR ribonuclease Csm6
    Article Snippet: .. The gene sequence of EiCsm6 was amplified from genomic DNA ( Enterococcus italicus DSM-15952) by PCR and cloned into a 1B vector (Addgene 29653) using ligation-independent cloning resulting in constructs carrying an N-terminal hexahistidine tag followed by a Tobacco Etch Virus (TEV) protease cleavage site. ..

    Article Title: Type III CRISPR-Cas systems generate cyclic oligoadenylate second messengers to activate Csm6 RNases
    Article Snippet: .. DNA sequences encoding for TtCsm6 ( Thermus thermophilus DSM-579), EiCsm6 ( Enterococcus italicus DSM-15952) and MtCsm6 ( Methanothermobacter thermoautotrophicus DSM-1053) were amplified from their respective genomic DNAs by a standard PCR protocol and were subsequently cloned into a 2HR-T vector (Addgene #29718) using ligation-independent cloning, resulting in constructs carrying an N-terminal hexahistidine tag followed by a StrepII tag, a Tobacco Etch Virus (TEV) protease cleavage site, and the Csm6 polypeptide sequence. .. To obtain a plasmid vector for heterologous expression a functional EiCsm(1-5) interference complex from the E. italicus DSM-15952 type III-A CRISPR locus, a DNA fragment spanning from the cas10 gene to the end of the second spacer (GenBank GL622241.1 ; complement 1999065-1991480) was amplified from E. italicus genomic DNA using primers oCGD208 and oCGD209 and cloned into a p7XNH3 vector using FX cloning , resulting in the pCGD253 plasmid.

    Article Title: Type III CRISPR-Cas systems produce cyclic oligoadenylate second messengers.
    Article Snippet: .. DNA sequences encoding for TtCsm6 (Thermus thermophilus DSM-579), EiCsm6 (Enterococcus italicus DSM-15952) and MtCsm6 (Methanothermobacter thermoautotrophicus DSM-1053) were amplified from their respective genomic DNAs by a standard PCR protocol and were subsequently cloned into a 2HR-T vector (Addgene #29718) using ligation-independent cloning, resulting in constructs carrying an N-terminal hexa-histidine tag followed by a StrepII tag, a Tobacco Etch Virus (TEV) protease cleavage site, and the Csm6 polypeptide sequence. ..

    Article Title: Activation and self-inactivation mechanisms of the cyclic oligoadenylate-dependent CRISPR ribonuclease Csm6
    Article Snippet: .. The gene sequence of EiCsm6 was amplified from genomic DNA ( Enterococcus italicus DSM-15952) by PCR and cloned into a 1B vector (Addgene 29653) using ligation-independent cloning resulting in constructs carrying an N-terminal hexahistidine tag followed by a tobacco etch virus (TEV) protease cleavage site. ..

    Virus:

    Article Title: Activation and self-inactivation mechanisms of the cyclic oligoadenylate-dependent CRISPR ribonuclease Csm6
    Article Snippet: .. The gene sequence of EiCsm6 was amplified from genomic DNA ( Enterococcus italicus DSM-15952) by PCR and cloned into a 1B vector (Addgene 29653) using ligation-independent cloning resulting in constructs carrying an N-terminal hexahistidine tag followed by a Tobacco Etch Virus (TEV) protease cleavage site. ..

    Article Title: Type III CRISPR-Cas systems generate cyclic oligoadenylate second messengers to activate Csm6 RNases
    Article Snippet: .. DNA sequences encoding for TtCsm6 ( Thermus thermophilus DSM-579), EiCsm6 ( Enterococcus italicus DSM-15952) and MtCsm6 ( Methanothermobacter thermoautotrophicus DSM-1053) were amplified from their respective genomic DNAs by a standard PCR protocol and were subsequently cloned into a 2HR-T vector (Addgene #29718) using ligation-independent cloning, resulting in constructs carrying an N-terminal hexahistidine tag followed by a StrepII tag, a Tobacco Etch Virus (TEV) protease cleavage site, and the Csm6 polypeptide sequence. .. To obtain a plasmid vector for heterologous expression a functional EiCsm(1-5) interference complex from the E. italicus DSM-15952 type III-A CRISPR locus, a DNA fragment spanning from the cas10 gene to the end of the second spacer (GenBank GL622241.1 ; complement 1999065-1991480) was amplified from E. italicus genomic DNA using primers oCGD208 and oCGD209 and cloned into a p7XNH3 vector using FX cloning , resulting in the pCGD253 plasmid.

    Article Title: Type III CRISPR-Cas systems produce cyclic oligoadenylate second messengers.
    Article Snippet: .. DNA sequences encoding for TtCsm6 (Thermus thermophilus DSM-579), EiCsm6 (Enterococcus italicus DSM-15952) and MtCsm6 (Methanothermobacter thermoautotrophicus DSM-1053) were amplified from their respective genomic DNAs by a standard PCR protocol and were subsequently cloned into a 2HR-T vector (Addgene #29718) using ligation-independent cloning, resulting in constructs carrying an N-terminal hexa-histidine tag followed by a StrepII tag, a Tobacco Etch Virus (TEV) protease cleavage site, and the Csm6 polypeptide sequence. ..

    Article Title: Activation and self-inactivation mechanisms of the cyclic oligoadenylate-dependent CRISPR ribonuclease Csm6
    Article Snippet: .. The gene sequence of EiCsm6 was amplified from genomic DNA ( Enterococcus italicus DSM-15952) by PCR and cloned into a 1B vector (Addgene 29653) using ligation-independent cloning resulting in constructs carrying an N-terminal hexahistidine tag followed by a tobacco etch virus (TEV) protease cleavage site. ..

    Expressing:

    Article Title: Accelerated RNA detection using tandem CRISPR nucleases.
    Article Snippet: The plasmid used to express MBP-tagged LbuCas13a (p2CT-His-MBP-Lbu_ C2c2_WT) is available from Addgene (83482). .. Plasmids used for expression of SUMO-tagged LbuCas13a (pGJK_His-SUMO-LbuCas13a), His-tagged EiCsm6 (pET28a_His-TEV-EiCsm6) and His-SUMO-tagged TtCsm6 (pET_ His6-SUMO-TEV-TtCsm6) will be available from Addgene (172488, 172487 and 172486). ..



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    a , b Overall structure of EiCsm6 homodimer bound to <t>cFA6,</t> shown in two orientations ( a : side view of the EiCsm6 homodimer; b : view of the cFA6 binding site at the CARF domain interface). The EiCsm6 homodimer is shown in cartoon representation (with protomers colored light and dark blue). cFA6 is depicted in sphere format. c Zoom-in view of the cA6 binding site at the CARF domain interface of EiCsm6, showing a 2m F O –D F C composite omit map, contoured at 1.0 σ and displayed within a radius of 2.2 Å around cFA6.
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    Image Search Results


    a , b Overall structure of EiCsm6 homodimer bound to cFA6, shown in two orientations ( a : side view of the EiCsm6 homodimer; b : view of the cFA6 binding site at the CARF domain interface). The EiCsm6 homodimer is shown in cartoon representation (with protomers colored light and dark blue). cFA6 is depicted in sphere format. c Zoom-in view of the cA6 binding site at the CARF domain interface of EiCsm6, showing a 2m F O –D F C composite omit map, contoured at 1.0 σ and displayed within a radius of 2.2 Å around cFA6.

    Journal: Nature Communications

    Article Title: Activation and self-inactivation mechanisms of the cyclic oligoadenylate-dependent CRISPR ribonuclease Csm6

    doi: 10.1038/s41467-020-15334-5

    Figure Lengend Snippet: a , b Overall structure of EiCsm6 homodimer bound to cFA6, shown in two orientations ( a : side view of the EiCsm6 homodimer; b : view of the cFA6 binding site at the CARF domain interface). The EiCsm6 homodimer is shown in cartoon representation (with protomers colored light and dark blue). cFA6 is depicted in sphere format. c Zoom-in view of the cA6 binding site at the CARF domain interface of EiCsm6, showing a 2m F O –D F C composite omit map, contoured at 1.0 σ and displayed within a radius of 2.2 Å around cFA6.

    Article Snippet: EiCsm6 crystals bound to cFA6 (synthesized by Biolog Life Science Institute GmbH & Co. KG) were grown in drops containing 120 μM EiCsm6 and 288 μM cFA6 equilibrating against a reservoir solution containing 100 mM HEPES pH 7.5, 1.6 M ammonium sulfate and 25 mM lithium perchlorate.

    Techniques: Binding Assay

    a – c Protein-cFA6 interactions. EiCsm6 is depicted in cartoon representation; cFA6 is shown as sticks. Hydrogen-bonding interactions are represented as black dotted lines. d Fluorogenic RNase activity assay of CARF domain mutants of EiCsm6 (0.5 nM) in the presence of 10 nM cyclic-hexa-AMP (cA6). AU, arbitrary units. e Growth curves (optical density at a wavelength of 600 nm) of Staphylococcus aureus strains harboring pTarget and pCRISPR plasmids. The pCRISPR plasmid expresses wild-type or CARF domain mutants of EiCsm6 and the Staphylococcus epidermidis Cas10–Csm effector complex containing an inactivating point mutation in the HD domain of Cas10. Data points in d , e represent the mean of three replicates; error bars represent the standard error of the mean (s.e.m.). Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: Activation and self-inactivation mechanisms of the cyclic oligoadenylate-dependent CRISPR ribonuclease Csm6

    doi: 10.1038/s41467-020-15334-5

    Figure Lengend Snippet: a – c Protein-cFA6 interactions. EiCsm6 is depicted in cartoon representation; cFA6 is shown as sticks. Hydrogen-bonding interactions are represented as black dotted lines. d Fluorogenic RNase activity assay of CARF domain mutants of EiCsm6 (0.5 nM) in the presence of 10 nM cyclic-hexa-AMP (cA6). AU, arbitrary units. e Growth curves (optical density at a wavelength of 600 nm) of Staphylococcus aureus strains harboring pTarget and pCRISPR plasmids. The pCRISPR plasmid expresses wild-type or CARF domain mutants of EiCsm6 and the Staphylococcus epidermidis Cas10–Csm effector complex containing an inactivating point mutation in the HD domain of Cas10. Data points in d , e represent the mean of three replicates; error bars represent the standard error of the mean (s.e.m.). Source data are provided as a Source Data file.

    Article Snippet: EiCsm6 crystals bound to cFA6 (synthesized by Biolog Life Science Institute GmbH & Co. KG) were grown in drops containing 120 μM EiCsm6 and 288 μM cFA6 equilibrating against a reservoir solution containing 100 mM HEPES pH 7.5, 1.6 M ammonium sulfate and 25 mM lithium perchlorate.

    Techniques: Activity Assay, Plasmid Preparation, Mutagenesis

    a Zoom-in view of nucleotides A1 and A6 of cFA6 bound to the EiCsm6 CARF domain. Residues implicated in the cleavage are represented as sticks; hydrogen-bonding interactions are depicted as black dotted lines. The red arrows represent the proposed reaction mechanism of cA6 cleavage involving nucleophilic attack of the 2’-hydroxyl group of A6 onto the phosphodiester group connecting A6 and A1. b RNase activity assay comparing the activity of WT and T11A EiCsm6 proteins in the presence of 500 nM cA6. The black arrows represent spike-ins of 0.8 pmol of RNaseAlert substrate at 5 min intervals. AU, arbitrary units. c Growth curves (optical density at a wavelength of 600 nm) of Staphylococcus aureus strains harboring pTarget and pCRISPR plasmids. The pCRISPR plasmid expresses wild-type or CARF domain mutants of EiCsm6 and the Staphylococcus epidermidis Cas10–Csm effector complex containing an inactivating point mutation in the Csm3 subunit (D32A; dCsm3). Data points in b , c represent the mean of three replicates; error bars represent the standard error of the mean (s.e.m.). Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: Activation and self-inactivation mechanisms of the cyclic oligoadenylate-dependent CRISPR ribonuclease Csm6

    doi: 10.1038/s41467-020-15334-5

    Figure Lengend Snippet: a Zoom-in view of nucleotides A1 and A6 of cFA6 bound to the EiCsm6 CARF domain. Residues implicated in the cleavage are represented as sticks; hydrogen-bonding interactions are depicted as black dotted lines. The red arrows represent the proposed reaction mechanism of cA6 cleavage involving nucleophilic attack of the 2’-hydroxyl group of A6 onto the phosphodiester group connecting A6 and A1. b RNase activity assay comparing the activity of WT and T11A EiCsm6 proteins in the presence of 500 nM cA6. The black arrows represent spike-ins of 0.8 pmol of RNaseAlert substrate at 5 min intervals. AU, arbitrary units. c Growth curves (optical density at a wavelength of 600 nm) of Staphylococcus aureus strains harboring pTarget and pCRISPR plasmids. The pCRISPR plasmid expresses wild-type or CARF domain mutants of EiCsm6 and the Staphylococcus epidermidis Cas10–Csm effector complex containing an inactivating point mutation in the Csm3 subunit (D32A; dCsm3). Data points in b , c represent the mean of three replicates; error bars represent the standard error of the mean (s.e.m.). Source data are provided as a Source Data file.

    Article Snippet: EiCsm6 crystals bound to cFA6 (synthesized by Biolog Life Science Institute GmbH & Co. KG) were grown in drops containing 120 μM EiCsm6 and 288 μM cFA6 equilibrating against a reservoir solution containing 100 mM HEPES pH 7.5, 1.6 M ammonium sulfate and 25 mM lithium perchlorate.

    Techniques: Activity Assay, Plasmid Preparation, Mutagenesis