CAS12B enzymes and systems
AssigneesThe Broad Institute, Inc., Massachusetts Institute of Technology
InventorsFeng Zhang, Jonathan Strecker, Ian Slaymaker, Sara Jones
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Abstract
Described herein are systems, methods, and compositions for targeting nucleic acids. In certain example embodiments, non-naturally occurring or engineered RNA-targeting systems contain an RNA-targeting Cas12b effector protein and at least one targeting nucleic acid component like a guide RNA or crRNA.
Claims26
A non-naturally occurring or engineered system comprising:
- i) a Cas12b effector protein, or a polynucleotide encoding the Cas12b effector protein, wherein the Cas12b effector is at least 95% identical to SEQ ID NO:
- 519;
- and
- ii) one or more guide molecules each comprising a guide sequence capable of hybridizing to a target sequence, and a tracrRNA, or a polynucleotide encoding the one or more guide molecules, and
- wherein the Cas12b effector protein comprises one or more mutations at amino acid positions selected from K846, S893, E837, or any combination thereof relative to SEQ ID NO:
- 519.
An isolated Cas12b effector protein at least 95% identical to SEQ ID NO:
- 519, wherein the Cas12b effector protein comprises one or more mutations at amino acid positions selected from K846, S893, E837, or any combination thereof relative to SEQ ID NO:
- 519,
- or an isolated nucleic acid encoding said Cas12b effector protein, optionally wherein the isolated nucleic acid is a DNA and further comprises a sequence encoding a crRNA and a tracr RNA.
An isolated eukaryotic cell comprising:
- i) a Cas12b effector protein, or a polynucleotide encoding the Cas12b effector protein, wherein the Cas12b effector protein is at least 95% identical to SEQ ID NO:
- 519;
- and
- ii) one or more guide molecules each comprising a guide sequence capable of hybridizing to a target sequence, and a tracrRNA, or one or more polynucleotides encoding the one or more guide molecules, and
- wherein the Cas12b effector protein comprises one or more mutations at amino acid positions selected from K846, S893, E837, or any combination thereof relative to SEQ ID NO:
- 519.
An engineered composition for site directed base editing comprising:
- (a) a targeting domain;
- (b) an adenosine deaminase, cytidine deaminase, or catalytic domain thereof;
- and (c) a guide molecule, wherein the targeting domain comprises a Cas12b effector protein or a polynucleotide encoding the Cas12b effector protein wherein the Cas12b effector protein is at least 95% identical to SEQ ID NO:
- 519,
- wherein the Cas12b effector protein comprises one or more mutations at amino acid positions selected from K846, S893, E837, or any combination thereof relative to SEQ ID NO:
- 519, and
- wherein said adenosine or cytidine deaminase protein or catalytic domain thereof is covalently or non-covalently linked to said Cas12b effector protein.
A system for detecting the presence of target polypeptides in one or more in vitro samples comprising:
- a Cas12b effector protein that is at least 95% identical to SEQ ID NO:
- 519, or a polynucleotide encoding the Cas12b effector protein, and
- wherein the Cas12b effector protein comprises one or more mutations at amino acid positions selected from K846, S893, E837, or any combination thereof relative to SEQ ID NO:
- 519;
- one or more detection aptamers, each designed to bind to one of the target polypeptides, each detection aptamer comprising a masked promoter binding site or masked primer binding site and a target sequence template;
- an oligonucleotide-based masking construct comprising a non-target sequence, further comprising nucleic acid amplification reagents to amplify a target sequence, and
- wherein the nucleic acid amplification reagents are isothermal amplification reagents.
Dependencies are inferred from the claim text.