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    Applications of Streptococcus-derived Cas9 nucleases on minimal Adenine-rich PAM targets
    2.
    发明申请
    Applications of Streptococcus-derived Cas9 nucleases on minimal Adenine-rich PAM targets 有权

    公开(公告)号：US20250154513A1

    公开(公告)日：2025-05-15

    申请号：US18927790

    申请日：2024-10-25

    Applicant: Massachusetts Institute of Technology

    Inventor： Pranam Chatterjee ,  Noah Michael Jakimo ,  Joseph M. Jacobson

    IPC: C12N15/74 ,  C12N9/22 ,  C12N15/11

    Abstract: Applications of a Streptococcus Cas9 ortholog from Streptococcus macacae (Smac Cas9), possessing minimal adenine-rich PAM specificity, include an isolated Streptococcus macacae Cas9 protein or transgene expression thereof, a CRISPR-associated DNA endonuclease with PAM interacting domain amino acid sequences that are at least 80% identical to that of the isolated Streptococcus macacae Cas9 protein, and an isolated, engineered Streptococcus pyogenes Cas9 (Spy Cas9) protein with a PID as either the PID amino acid composition of the isolated Streptococcus macacae Cas9 (Smac Cas9) protein or of a CRISPR-associated DNA endonuclease with PID amino acid sequences that are at least 80% identical to that of the isolated Streptococcus macacae Cas9 protein. A method for altering expression of at least one gene product employs Streptococcus macacae Cas9 endonucleases in complex with guide RNA, for specific recognition and activity on a DNA target immediately upstream of either an “NAA” or “NA” or “NAAN” PAM sequence.

    Applications of engineered Streptococcus canis Cas9 variants on single-base PAM targets
    3.
    发明授权
    Applications of engineered Streptococcus canis Cas9 variants on single-base PAM targets 有权

    公开(公告)号：US11453865B2

    公开(公告)日：2022-09-27

    申请号：US16689071

    申请日：2019-11-19

    Applicant: Massachusetts Institute of Technology

    Inventor： Pranam Chatterjee ,  Noah Michael Jakimo ,  Joseph M. Jacobson

    IPC: C12N9/22 ,  C12N15/11 ,  C12N15/85

    Abstract: Engineered Streptococcus canis Cas9 (ScCas9) variants include an ScCas9 protein with its PID being the PID amino acid composition of Streptococcus pyogenes Cas9 (SpCas9)-NG, an ScCas9 protein having a threonine-to-lysine substitution mutation at position 1227 in its amino acid sequence (Sc+), and an ScCas9 protein having a threonine-to-lysine substitution mutation at position 1227 and a substitution of residues ADKKLRKRSGKLATE [SEQ ID No. 4] in position 365-379 in the ScCas9 open reading frame (Sc++). Also included are CRISPR-associated DNA endonucleases with a PAM specificity of 5′-NG-3′ or 5′-NNG-3′ and a method of altering expression of a gene product by utilizing the engineered ScCas9 variants.

    Applications of Streptococcus-derived Cas9 nucleases on minimal Adenine-rich PAM targets
    4.
    发明申请
    Applications of Streptococcus-derived Cas9 nucleases on minimal Adenine-rich PAM targets 有权

    公开(公告)号：US20220162620A1

    公开(公告)日：2022-05-26

    申请号：US17053713

    申请日：2019-05-06

    Applicant: Massachusetts Institute of Technology

    Inventor： Pranam Chatterjee ,  Noah Michael Jakimo ,  Joseph M Jacobson

    IPC: C12N15/74 ,  C12N9/22 ,  C12N15/11

    Abstract: Applications of a Streptococcus Cas9 ortholog from Streptococcus macacae (Smac Cas9), possessing minimal adenine-rich PAM specificity, include an isolated Streptococcus macacae Cas9 protein or transgene expression thereof, a CRISPR-associated DNA endonuclease with PAM interacting domain amino acid sequences that are at least 80% identical to that of the isolated Streptococcus macacae Cas9 protein, and an isolated, engineered Streptococcus pyogenes Cas9 (Spy Cas9) protein with a PID as either the PID amino acid composition of the isolated Streptococcus macacae Cas9 (Smac Cas9) protein or of a CRISPR-associated DNA endonuclease with PID amino acid sequences that are at least 80% identical to that of the isolated Streptococcus macacae Cas9 protein. A method for altering expression of at least one gene product employs Streptococcus macacae Cas9 endonucleases in complex with guide RNA, for specific recognition and activity on a DNA target immediately upstream of either an “NAA” or “NA” or “NAAN” PAM sequence.

    Applications of Engineered Streptococcus Canis Cas9 Variants on Single-Base PAM Targets
    6.
    发明申请
    Applications of Engineered Streptococcus Canis Cas9 Variants on Single-Base PAM Targets 审中-公开

    公开(公告)号：US20200332271A1

    公开(公告)日：2020-10-22

    申请号：US16689071

    申请日：2019-11-19

    Applicant: Massachusetts Institute of Technology

    Inventor： Pranam Chatterjee ,  Noah Michael Jakimo ,  Joseph M. Jacobson

    IPC: C12N9/22 ,  C12N15/11 ,  C12N15/85

    Abstract: Engineered Streptococcus canis Cas9 (ScCas9) variants include an ScCas9 protein with its PID being the PID amino acid composition of Streptococcus pyogenes Cas9 (SpCas9)-NG, an ScCas9 protein having a threonine-to-lysine substitution mutation at position 1227 in its amino acid sequence (Sc+), and an ScCas9 protein having a threonine-to-lysine substitution mutation at position 1227 and a substitution of residues ADKKLRKRSGKLATE in position 365-379 in the ScCas9 open reading frame (Sc++). Also included are CRISPR-associated DNA endonucleases with a PAM specificity of 5′-NG-3′ or 5′-NNG-3′ and a method of altering expression of a gene product by utilizing the engineered ScCas9 variants.

    Chimeric DNA:RNA Guide for High Accuracy Cas9 Genome Editing
    8.
    发明申请
    Chimeric DNA:RNA Guide for High Accuracy Cas9 Genome Editing 审中-公开

    公开(公告)号：US20180282722A1

    公开(公告)日：2018-10-04

    申请号：US15820425

    申请日：2017-11-21

    Applicant: Massachusetts Institute of Technology

    Inventor： Noah Jakimo ,  Pranam Chatterjee ,  Joseph M. Jacobson

    IPC: C12N15/11 ,  C12N9/22 ,  C12N9/96 ,  C12N15/90

    Abstract: A chimeric DNA:RNA guide for very high accuracy Cas9 genome editing employs nucleotide-type substitutions in nucleic acid-guided endonucleases for enhanced specificity. The CRISPR-Cas9 gene editing system is manipulated to generate chimeric DNA:RNA guide strands to minimize the off-target cleavage events of the S. pyogenes Cas9 endonuclease. A DNA:RNA chimeric guide strand is sufficient to guide Cas9 to a specified target sequence for indel formation and minimize off-target cleavage events due to the specificity conferred by DNA-DNA interactions. Use of chimeric mismatch-evading lowered-thermostability guides (“melt-guides”) demonstrate that nucleotide-type substitutions in the spacer can reduce cleavage of sequences mismatched by as few as a single base pair. The chimeric mismatch-evading lowered-thermostability guides replace most gRNA spacer positions with DNA bases to suppress mismatched targets under Cas9's catalytic threshold.

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