Crispr-associated transposase systems and methods of use thereof

Abstract

The present application provides systems, methods and compositions used for targeted gene modification, targeted insertion, perturbation of gene transcripts, nucleic acid editing. Novel nucleic acid targeting systems comprise components of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems and transposable elements.

1 - 28 . (canceled) 
     
     
         29 . A delivery system configured to deliver one or more components of an engineered nucleic acid targeting system comprising:
 (a) one or more CRISPR-associated Tn7 transposase or Tn7-like transposase proteins that form a transposase;   (b) a catalytically inactive Type V Cas protein capable of forming a complex with the transposase;   (c) a guide molecule capable of forming a complex with the Type V Cas protein and directing sequence-specific binding of a guide-Cas protein complex to a target sequence of a target polynucleotide; and   (d) a donor construct, comprising a heterologous donor polynucleotide sequence flanked by a first and second binding sequence capable of complexing the donor construct with the one or more Tn7 or Tn7-like transposase proteins.   
     
     
         30 . The delivery system of  claim 29 , further comprising one or more vectors. 
     
     
         31 . The delivery system of  claim 30 , wherein the one or more vectors comprise one or more polynucleotides encoding components (a), (b), and (c). 
     
     
         32 . The delivery system of  claim 31 , wherein components (a), (b), and (c) are on the same vector. 
     
     
         33 . The delivery system of  claim 31 , wherein components (a), (b), and (c) are on different vectors. 
     
     
         34 . The delivery system of  claim 31 , wherein components (a) and (b) are on a first vector and component (c) is on a second vector. 
     
     
         35 . The delivery system of  claim 31 , wherein component (a) is on a first vector and components (b) and (c) are on a second vector. 
     
     
         36 . The delivery system of  claim 31 , wherein the one or more polynucleotides are codon optimized for expression in a eukaryotic cell. 
     
     
         37 . The delivery system of  claim 30 , wherein the one or more vectors comprise one or more viral vectors further comprising one or more polynucleotides encoding components (a), (b), and (c). 
     
     
         38 . The delivery system of  claim 37 , wherein the one or more viral vectors comprise retroviral, lentiviral, adenoviral, adeno-associated, or herpes simplex viral vectors. 
     
     
         39 . The delivery system of  claim 31 , wherein component (a) comprises (i) TnsB and TnsC, or (ii) TniA and TniB. 
     
     
         40 . The delivery system of  claim 39 , wherein TnsB and TnsC are on the same vector or on different vectors. 
     
     
         41 . The delivery system of  claim 40 , wherein components (b) and (c), TnsB, and TnsC are on the same vector. 
     
     
         42 . The delivery system of  claim 40 , wherein components (b) and (c), and either TnsB or TnsC are on the same vector. 
     
     
         43 . The delivery system of  claim 39 , wherein TniA and TniB are on the same vector or on different vectors. 
     
     
         44 . The delivery system of  claim 43 , wherein components (b) and (c), TniA, and TniB are on the same vector. 
     
     
         45 . The delivery system of  claim 43 , wherein components (b) and (c), and either TniA or TniB are on the same vector. 
     
     
         46 . The delivery system of  claim 31 , wherein component (a) comprises:
 (i) TnsA, TnsB, TnsC, and TniQ;   (ii) TnsA, TnsB, and TnsC;   (iii) TnsB, TnsC, and TniQ;   (iv) TnsA, TnsB, and TniQ;   (v) TnsE;   (vi) TniA, TniB, and TniQ;   (vii) TnsB, TnsC, and TnsD;   (viii) TnpA; or   (ix) any combination thereof.   
     
     
         47 . The delivery system of  claim 46 , wherein one or more of subcomponents (i)-(ix) are on the same vector or on different vectors. 
     
     
         48 . The delivery system of  claim 31 , wherein component (a) comprises TnsB, TnsC, and TniQ. 
     
     
         49 . The delivery system of  claim 43 , wherein TnsB, TnsC, and TniQ are on the same vector or on different vectors. 
     
     
         50 . The delivery system of  claim 49 , wherein components (b) and (c), and one or more of TnsB, TnsC, and TniQ are on the same vector. 
     
     
         51 . The delivery system of  claim 48 , wherein the TnsB, TnsC, and TniQ are proteins selected from Table 28 or Table 29, or are encoded by polynucleotides selected from Table 26 or Table 27. 
     
     
         52 . The delivery system of  claim 29 , wherein the Type V Cas protein comprises Cas12k. 
     
     
         53 . The delivery system of  claim 51 , wherein the Cas12k is encoded by a polynucleotide selected from Table 26 or Table 27; or is a protein selected from Table 28 or Table 29; or is selected from an organism of  FIGS.  2 A  and 2B or Table 2. 
     
     
         54 . The delivery system of  claim 29 , wherein the guide molecule comprises a polynucleotide sequence selected from Table 26.