Small type ii-d cas proteins and methods of use thereof

Abstract

Described herein are systems, methods, and compositions capable of targeting nucleic acids. Describe in certain exemplary embodiments herein are a class of small Cas proteins (Type II-D Cas proteins) and systems thereof. Also described in certain exemplary embodiments herein are methods of modifying target sequences using the class of small Cas proteins (Type II-D Cas proteins) and systems thereof described herein.

What is claimed is: 
     
         1 . An engineered nucleic acid targeting system comprising:
 a Cas protein comprising a RuvC domain and an HNH domain, wherein the Cas protein is about 950 amino acids or less in size, wherein the Cas protein is capable of forming a complex with a nucleic acid guide molecule, and wherein the nucleic acid guide molecule is capable of sequence-specific binding of a target nucleic acid sequence on a target polynucleotide.   
     
     
         2 . The engineered nucleic acid targeting system of  claim 1 , wherein the Cas protein is less than or equal to 780 amino acids in size. 
     
     
         3 . The engineered nucleic acid targeting system of any one of  claims 1 - 2 , wherein the Cas protein has no association with Cas1, Cas2, Cas4, or Csn2. 
     
     
         4 . The engineered nucleic acid targeting system of any one of  claims 1 - 3 , wherein the Cas protein is a Type II Cas protein. 
     
     
         5 . The engineered nucleic acid targeting system of  claim 4 , wherein the Type II Cas protein is a Type II-D Cas protein. 
     
     
         6 . The engineered nucleic acid targeting system of any one of  claims 1 - 5 , wherein the Cas protein is encoded at least in part or in a whole by polynucleotide sequence that is about 70-100 percent identical to any one of SEQ ID NOs: 31-133 or a portion thereof. 
     
     
         7 . The engineered nucleic acid targeting system of any one of  claims 1 - 6 , wherein the Cas protein is from or originated from  Gammaproteobacteria bacterium  AqS3 , Deltaproteobacteria bacterium  GWF2_42_12, JGI Metagenome: IMG 3300025323 , Nitrospirae bacterium  RBG_13_39_12, or  Nitrospiraceae bacterium  isolate UBA9935. 
     
     
         8 . The engineered nucleic acid targeting system of any one of  claims 1 - 6 , wherein the Cas protein is capable of forming a complex with two or more nucleic guide molecules, wherein each guide molecule is capable of sequence-specific binding of a target nucleic acid sequence, wherein each target sequence is different. 
     
     
         9 . The engineered nucleic acid targeting system of  claim 8 , wherein the target sequences are on the same or are on different target polynucleotides. 
     
     
         10 . The engineered nucleic acid targeting system of any one of  claims 1 - 9 , wherein the guide molecule or the two or more guide molecules are capable of sequence-specific binding a target sequence in vitro, in situ, ex vivo, or in vivo. 
     
     
         11 . The engineered nucleic acid targeting system of any one of  claims 1 - 10 , wherein the guide molecule or the two or more guide molecules are capable of sequence-specific binding a target sequence in a prokaryotic cell, eukaryotic cell, a virus, or a combination thereof. 
     
     
         12 . The engineered nucleic acid targeting system of any one of  claims 1 - 11 , wherein the Cas protein is operably coupled to one or more nuclear localization signals. 
     
     
         13 . The engineered nucleic acid targeting system of any one of  claims 1 - 12 , wherein the Cas protein is operably coupled to one or more nuclear export signals. 
     
     
         14 . The engineered nucleic acid targeting system of any one of  claims 1 - 13 , wherein the Cas protein lacks one or more catalytic activates. 
     
     
         15 . The engineered nucleic acid targeting system of any one of  claims 1 - 14 , wherein the Cas protein lacks nuclease activity. 
     
     
         16 . The engineered nucleic acid targeting system of any one of  claims 1 - 16 , wherein the Cas protein is a nickase. 
     
     
         17 . The engineered nucleic acid targeting system of any one of  claims 1 - 16 , wherein the Cas protein is operably coupled to or associated with one or more functional domains. 
     
     
         18 . The engineered nucleic acid targeting system of  claim 17 , wherein the one or more functional domains is/are one or more heterologous functional domains. 
     
     
         19 . The engineered nucleic acid targeting system of any one of  claims 17 - 18 , wherein the one or more functional domains has one or more activities selected from deaminase activity, methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, nuclease activity, single-strand RNA cleavage activity, double-strand RNA cleavage activity, single-strand DNA cleavage activity, double-strand DNA cleavage activity, nucleic acid binding activity, transposition activity, reverse transcription activity, or a combination thereof. 
     
     
         20 . The engineered nucleic acid targeting system of any one of  claims 17 - 19 , wherein the one or more functional domains is capable of cleaving the target polynucleotide. 
     
     
         21 . The engineered nucleic acid targeting system of any one of  claims 17 - 20 , wherein the one or more functional domains is capable of modifying transcription or translation of the target polynucleotide. 
     
     
         22 . The engineered nucleic acid targeting system of any one of  claims 1 - 21 , further comprising a recombination template. 
     
     
         23 . The engineered nucleic acid targeting system of  claim 22 , wherein the recombination template is operably coupled to, complexed with, or is associated with the Cas protein, the nucleic acid guide molecule, or both. 
     
     
         24 . The engineered nucleic acid targeting system of any one of  claims 22 - 23 , wherein the recombination template is a homology-directed repair (HDR) recombination template. 
     
     
         25 . The engineered nucleic acid targeting system of any one of  claims 1 - 24 , wherein the nucleic acid targeting system comprises a tracrRNA. 
     
     
         26 . The engineered nucleic acid targeting system of any one of  claims 1 - 25 , wherein the Cas protein is a chimeric protein comprising a first polypeptide fragment from a first Cas protein and a second polypeptide fragment from a second Cas protein. 
     
     
         27 . The engineered nucleic acid targeting system of any one of  claims 1 - 25 , further comprising a deaminase or catalytic domain thereof. 
     
     
         28 . The engineered nucleic acid targeting system of  claim 27 , wherein the deaminase is an adenosine deaminase or a cytidine deaminase. 
     
     
         29 . The engineered nucleic acid targeting system of any one of  claims 27 - 28 , wherein the deaminase or catalytic domain thereof is operably coupled to, complexed with, or otherwise associated with the Cas protein, a guide molecule, or both or is capable of operably coupling to, complexing with, or otherwise associated with the Cas protein, a guide molecule, or both after delivery to a cell. 
     
     
         30 . The engineered nucleic acid targeting system of any one of  claims 27 - 29 , wherein the nucleotide deaminase or catalytic domain thereof has been modified to increase its activity against a DNA-RNA heteroduplex, to reduce off-target effects, or both. 
     
     
         31 . The engineered nucleic acid targeting system of any one of  claims 1 - 30 , further comprising a reverse transcriptase or functional domain thereof, wherein the reverse transcriptase or functional domain thereof is optionally operably coupled to, is capable of complexing with, or is otherwise associated with the Cas protein, the guide molecule, or both. 
     
     
         32 . The engineered nucleic acid targeting system of any one of  claims 1 - 31 , further comprising one or more nucleic acid guide molecules, wherein each of the one or more nucleic acid guide molecules is capable of capable of forming a complex or is complexed with the Cas protein, and wherein each of the one or more nucleic acid guide molecules is capable of sequence specific binding of a target sequence in a target polynucleotide. 
     
     
         33 . The engineered nucleic acid targeting system of any one of  claims 1 - 32 , wherein the engineered nucleic acid targeting system is capable of modifying a sequence of the target polynucleotide. 
     
     
         34 . The engineered nucleic acid targeting system of  claim 33 , wherein the modification is
 a. insertion of one or more polynucleotides;   b. deletion of one or more polynucleotides;   c. conversion of a C•G base pair to a T•A base pair;   d. conversion of an A•T base pair to a G•C base pair; or   e. a combination thereof.   
     
     
         35 . The engineered nucleic acid targeting system of any one of  claims 32 - 34 , wherein the modification alters a transcription product of the target polynucleotide, a translation product of the target polynucleotide, or both. 
     
     
         36 . The engineered nucleic acid targeting system of any one of  claims 32 - 35 , wherein the modification alters transcription, translation, or both of the target polynucleotide. 
     
     
         37 . A polynucleotide comprising one or more nucleic acid sequences that encode one or more components of the engineered nucleic acid system of any one of  claims 1 - 36 . 
     
     
         38 . The polynucleotide of  claim 37 , wherein the polynucleotide is codon optimized for expression in a eukaryotic cell. 
     
     
         39 . The polynucleotide of  claim 38 , wherein the eukaryotic cell is a human cell or a non-human animal cell. 
     
     
         40 . A vector system comprising:
 one or more vectors comprising one or more polynucleotides of any one of  claims 37 - 39 , and optionally one or more regulatory elements operably coupled to one or more polynucleotides.   
     
     
         41 . The vector system of  claim 40 , wherein the one or more of the one or more vectors are viral vectors. 
     
     
         42 . The vector system of  claim 41 , wherein the viral vector(s) is/are a retroviral vector(s), lentiviral vector(s), adenoviral vector(s), adeno-associated viral vector(s), herpes simplex viral vector(s), or a combination thereof. 
     
     
         43 . A delivery composition comprising:
 a. an engineered nucleic acid targeting system of any one of  claims 1 - 36 ;   b. one or more polynucleotides of any one of  claims 37 - 39 ;   c. one or more vector systems of any one of  claims 40 - 42 ; or   d. a combination thereof; and
 a delivery vehicle, wherein a, b, c, d, or e, are associated with or operably coupled to the delivery vehicle. 
   
     
     
         44 . A cell or progeny thereof comprising:
 a. an engineered nucleic acid targeting system of any one of  claims 1 - 36 ;   b. one or more polynucleotides of any one of  claims 37 - 39 ;   c. one or more vector systems of any one of  claims 40 - 42 ;   d. a delivery formulation as in  claim 43 ;   e. one or more polynucleotide modifications produced by an engineered nucleic acid targeting system of any one of  claims 1 - 36 ; or   f. a combination thereof.   
     
     
         45 . The cell or progeny thereof of  claim 44 , wherein the cell or progeny thereof is a prokaryotic or eukaryotic cell. 
     
     
         46 . A tissue, organ, or organism comprising:
 a cell or progeny thereof as in any one of  claims 44 - 45  or a population thereof.   
     
     
         47 . A pharmaceutical formulation comprising:
 a. an engineered nucleic acid targeting system of any one of  claims 1 - 36 ;   b. one or more polynucleotides of  claim 37 - 39 ;   c. one or more vector systems of any one of  claims 40 - 42 ;   d. a delivery formulation as in  claim 43 ;   e. a cell or progeny thereof as in any one of  claims 44 - 45 ;   f. a tissue, an organ, or an organism as in  claim 46 ; or   g. a combination thereof; and
 a pharmaceutically acceptable carrier. 
   
     
     
         48 . A product produced by a cell or progeny thereof as in any one of  claims 44 - 45  or a population thereof, a tissue, organ, or organism as in  claim 46 , or both. 
     
     
         49 . A method of modifying one or more target polynucleotides, the method comprising contacting the one or more target polynucleotides with an engineered nucleic acid targeting system of any one of  claims 1  to  36 , wherein the engineered nucleic acid targeting system is directed to the one or more target sequences by the guide nucleic acid guide molecule(s) of the engineered nucleic acid targeting system, whereby one or more target polynucleotides is/are modified. 
     
     
         50 . The method of  claim 49 , wherein the modification is
 a. insertion of one or more polynucleotides;   b. deletion of one or more polynucleotides;   c. conversion of a C•G base pair to a T•A base pair;   d. conversion of an A•T base pair to a G•C base pair; or   e. a combination thereof.   
     
     
         51 . The method of any one of  claims 49 - 50 , wherein contacting occurs in vitro, in situ, ex vivo, or in vivo. 
     
     
         52 . The method of any one of  claims 49 - 51 , wherein contacting occurs within a cell. 
     
     
         53 . A modified polynucleotide or modified cell or progeny thereof produced from a method as in any one of  claims 49 - 51 . 
     
     
         54 . A modified cell or progeny thereof as in  claim 53 , wherein the cell is a eukaryotic cell or progeny thereof. 
     
     
         55 . The modified cell or progeny thereof of  claim 54 , wherein the cell or progeny thereof is a human cell or progeny thereof or a non-human animal cell or progeny thereof. 
     
     
         56 . The modified cell or progeny thereof of  claim 54 , wherein the cell or progeny thereof is a plant cell. 
     
     
         57 . A method of treating and/or preventing a disease, condition, or a symptom thereof in a subject in need thereof, the method comprising:
 administering to the subject in need thereof
 a. an engineered nucleic acid targeting system of any one of  claims 1 - 36 ; 
 b. one or more polynucleotides of  claim 37 - 39 ; 
 c. one or more vector systems of any one of  claims 40 - 42 ; 
 d. a delivery formulation as in  claim 43 ; 
 e. a cell or progeny thereof as in any one of  claims 44 - 45 ; 
 f. a tissue, an organ, or an organism as in  claim 46 ; 
 g. a pharmaceutical formulation as in  claim 47 ; 
 h. a product as in  claim 48 ; or 
 i. any combination thereof. 
   
     
     
         58 . A method of treating and/or preventing a disease, condition, or a symptom thereof in a subject or cell thereof, the method comprising:
 modifying one or more target polynucleotides in or from the subject or cell thereof by contacting the one or more target polynucleotides with an engineered nucleic acid targeting system of any one of  claims 1  to  36 , wherein the engineered nucleic acid targeting system is directed to the one or more target sequences in one or more target polynucleotides by the guide nucleic acid guide molecule(s) of the engineered nucleic acid targeting system, whereby one or more target polynucleotides is/are modified.   
     
     
         59 . The method of  claim 58 , wherein contacting occurs in vitro, in situ, ex vivo, or in vivo. 
     
     
         60 . The method of  claim 59 , wherein contacting occurs ex vivo in a cell obtained from the subject or progeny thereof and wherein the method further comprises administering cell or obtained from the subject or progeny to the subject after contacting the cell or progeny thereof with the engineered targeting system. 
     
     
         61 . A method of generating a modified organism, the method comprising:
 modifying one or more target polynucleotides in a cell by a method as in any one of  claims 50 - 52 .   
     
     
         62 . The method of  claim 62 , wherein the organism is a non-human animal. 
     
     
         63 . The method of  claim 62 , wherein the organism is a plant. 
     
     
         64 . A method of identifying a trait of interest in an organism where the trait of interest is encoded by one or more target polynucleotides, the method comprising:
 contacting the organism or a sample therefrom comprising polynucleotides with an engineered nucleic acid targeting system of any one of  claims 1 - 36 , wherein the engineered nucleic acid targeting system is directed to the one or more target sequences by the guide nucleic acid guide molecule(s) of the engineered nucleic acid targeting system, whereby one or more target polynucleotides, and thereby the one or more traits, are identified.   
     
     
         65 . The method of  claim 65 , wherein one or more target polynucleotides are modified by the engineered nucleic acid targeting system. 
     
     
         66 . The method of any of  claims 65 - 66 , wherein the method is performed in vitro, in situ, ex vivo, or in vivo. 
     
     
         67 . The method of any of  claims 65 - 67 , wherein the organism is a plant, non-human animal, or human. 
     
     
         68 . A method of identifying a polynucleotide modifier, the method comprising:
 exposing one or more polynucleotides to one or more candidate agents; and   detecting one or more modified polynucleotides by contacting the one or more polynucleotides exposed to one or more candidate agents with an engineered nucleic acid targeting system of any one of  claims 1 - 36 , wherein the engineered nucleic acid targeting system is directed to the one or more target sequences of one or more modified target polynucleotides present in the sample by the guide nucleic acid guide molecule(s) of the engineered nucleic acid targeting system, whereby one or more modified target polynucleotides present in the sample are identified.   
     
     
         69 . A method of detecting one or more target polynucleotide present in a sample comprising polynucleotides, the method comprising:
 contacting, in vitro, one or more target polynucleotides present in the sample with an engineered nucleic acid targeting system of any one of  claims 1 - 36 , wherein the engineered nucleic acid targeting system is directed to the one or more target sequences of one or more target polynucleotides present in the sample by the guide nucleic acid guide molecule(s) of the engineered nucleic acid targeting system, whereby one or more target polynucleotides present in the sample are identified.