Crispr systems with engineered dual guide nucleic acids

Abstract

The present invention relates to an engineered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system comprising engineered dual guide nucleic acids (e.g., RNAs) capable of activating a CRISPR-Associated (Cas) nuclease, such as a type V-A Cas nuclease. Also provided are methods of targeting, editing, and/or modifying a nucleic acid using the engineered CRISPR system, and compositions and cells comprising the engineered CRISPR system.

1 - 45 . (canceled) 
     
     
         46 . An engineered, non-naturally occurring system comprising:
 (a) a targeter nucleic acid comprising:
 (i) a spacer sequence designed to hybridize with a target nucleotide sequence; and 
 (ii) a targeter stem sequence; and 
   (b) a modulator nucleic acid comprising a modulator stem sequence complementary to the targeter stem sequence,   wherein the targeter nucleic acid and the modulator nucleic acid are separate nucleic acids, and   wherein a complex comprising the targeter nucleic acid and the modulator nucleic acid is capable of activating a CRISPR Associated (Cas) nuclease that, in a naturally occurring system, is activated by a single crRNA in the absence of a tracrRNA, wherein the Cas nuclease comprises a Type V-A Cas nuclease; and   (c) the Type V-A Cas nuclease.   
     
     
         47 . The engineered, non-naturally occurring system of  claim 46  wherein the targeter stem sequence and the modulator stem sequence are each 4-10 nucleotides in length. 
     
     
         48 . The engineered, non-naturally occurring system of  claim 46  wherein the spacer sequence is 18 nucleotides in length or shorter. 
     
     
         49 . The engineered, non-naturally occurring system of  claim 46  wherein the targeter nucleic acid comprises, from 5′ to 3′, the targeter stem sequence, the spacer sequence, and an optional additional nucleotide sequence. 
     
     
         50 . The engineered, non-naturally occurring system of  claim 46  wherein the targeter nucleic acid, the modulator nucleic acid, or both, comprises a ribonucleic acid (RNA). 
     
     
         51 . The engineered, non-naturally occurring system of  claim 50  wherein the targeter nucleic acid, the modulator nucleic acid, or both, comprises a chemically modified RNA. 
     
     
         52 . The engineered, non-naturally occurring system of  claim 51  wherein the chemical modification is present in one or more nucleotides at the 3′ end of the targeter nucleic acid, in one or more nucleotides at the 5′ end of the modulator nucleic acid, or both. 
     
     
         53 . The engineered, non-naturally occurring system of  claim 52  wherein the chemical modification is selected from the group consisting of 2′-O-methyl, 2′-fluoro, 2′-O-methoxyethyl, phosphorothioate, phosphorodithioate, pseudouridine, and any combinations thereof. 
     
     
         54 . The engineered, non-naturally occurring system of  claim 46  wherein the modulator nucleic acid further comprises an additional nucleotide sequence positioned 5′ to the modulator stem sequence, and 4-50 nucleotides in length. 
     
     
         55 . The engineered, non-naturally occurring system of  claim 54  wherein the additional nucleotide sequence comprises a donor template-recruiting sequence capable of hybridizing with a donor template. 
     
     
         56 . The engineered, non-naturally occurring system of  claim 55  further comprising the donor template. 
     
     
         57 . The engineered, non-naturally occurring system of  claim 46  wherein the targeter nucleic acid and the modulator nucleic acid are not covalently linked. 
     
     
         58 . The engineered, non-naturally occurring system of  claim 46  wherein the Cas nuclease comprises an amino acid sequence at least 80% identical to SEQ ID NO: 1. 
     
     
         59 . The engineered, non-naturally occurring system of  claim 46  wherein the Cas nuclease comprises an amino acid sequence at least 95% identical to SEQ ID NO: 1. 
     
     
         60 . The engineered, non-naturally occurring system of  claim 46  wherein the targeter nucleic acid, the modulator nucleic acid, and the Cas nuclease are present in a ribonucleoprotein (RNP) complex. 
     
     
         61 . A cell comprising
 (a) a targeter nucleic acid, or a polynucleotide encoding thereof, comprising
 (i) a spacer sequence designed to hybridize with a target nucleotide sequence and 
 (ii) a targeter stem sequence; 
   (b) a modulator nucleic acid, or a polynucleotide encoding thereof, comprising a modulator stem sequence complementary to the targeter stem sequence,   wherein the targeter nucleic acid and the modulator nucleic acid are expressed as separate nucleic acids, and wherein a complex comprising the targeter nucleic acid and the modulator nucleic acid is capable of activating a Cas nuclease that, in a naturally occurring system, is activated by a single crRNA in the absence of a tracrRNA, wherein the Cas nuclease comprises a Type V-A nuclease; and   (c) a Type V-A Cas nuclease, or a polynucleotide encoding thereof.   
     
     
         62 . The cell of  claim 61  wherein the cell is an immune cell or a stem cell. 
     
     
         63 . The cell of  claim 61  wherein the cell is an induced pluripotent stem cell. 
     
     
         64 . The cell of  claim 61  wherein the cell is an immune cell that is a T lymphocyte. 
     
     
         65 . A method of cleaving a target DNA having a target nucleotide sequence, the method comprising contacting the target DNA with an engineered, non-naturally occurring system comprising
 (a) a targeter nucleic acid comprising:
 (i) a spacer sequence designed to hybridize with the target nucleotide sequence; and 
 (ii) a targeter stem sequence; and 
   (b) a modulator nucleic acid comprising a modulator stem sequence complementary to the targeter stem sequence,   wherein the targeter nucleic acid and the modulator nucleic acid are separate nucleic acids, and   wherein a complex comprising the targeter nucleic acid and the modulator nucleic acid is capable of activating a CRISPR Associated (Cas) nuclease that, in a naturally occurring system, is activated by a single crRNA in the absence of a tracrRNA, wherein the Cas nuclease comprises a type V-A Cas nuclease, and   (c) the Cas nuclease;   thereby resulting in cleavage of the target DNA.   
     
     
         66 . The method of  claim 65  wherein the contacting occurs in vitro. 
     
     
         67 . The method of  claim 65  wherein the contacting occurs in a cell ex vivo. 
     
     
         68 . The method of  claim 67  wherein the target DNA is genomic DNA of the cell. 
     
     
         69 . The method of  claim 67  wherein the system is delivered into the cell as a pre-formed RNP complex. 
     
     
         70 . The method of  claim 69  wherein the pre-formed RNP complex is delivered into the cell by electroporation. 
     
     
         71 . A method of editing a genome of a eukaryotic cell, the method comprising delivering an engineered, non-naturally occurring system comprising
 (a) a targeter nucleic acid comprising:
 (i) a spacer sequence designed to hybridize with a target nucleotide sequence within the genome; and 
 (ii) a targeter stem sequence; and 
   (b) a modulator nucleic acid comprising a modulator stem sequence complementary to the targeter stem sequence,   wherein the targeter nucleic acid and the modulator nucleic acid are separate nucleic acids, and   wherein a complex comprising the targeter nucleic acid and the modulator nucleic acid is capable of activating a CRISPR Associated (Cas) nuclease that, in a naturally occurring system, is activated by a single crRNA in the absence of a tracrRNA, wherein the Cas nuclease comprises a type V-A Cas nuclease, and   (c) the Cas nuclease; and/or a polynucleotide or polynucleotides coding for one or more of (a), (b), and/or (c),   into the eukaryotic cell, thereby resulting in editing of the genome of the eukaryotic cell.   
     
     
         72 . The method of  claim 71  wherein the system is delivered into the cell as a pre-formed RNP complex. 
     
     
         73 . The method of  claim 72  wherein the system is delivered into the cell by electroporation. 
     
     
         74 . The method of  claim 71  wherein the cell is an immune cell or a stem cell. 
     
     
         75 . The method of  claim 74  wherein the cell is an immune cell that is a T lymphocyte.