Altering microbial populations & modifying microbiota

Abstract

The invention relates to methods, uses, systems, arrays, engineered nucleotide sequences and vectors for inhibiting bacterial population growth or for altering the relative ratio of sub-populations of first and second bacteria in a mixed population of bacteria. The invention is particularly useful, for example, for treatment of microbes such as for environmental, medical, food and beverage use. The invention relates inter alia to methods of controlling microbiologically influenced corrosion (MIC) or biofouling of a substrate or fluid in an industrial or domestic system.

What is claimed is: 
     
         1 . A method for modifying a target DNA sequence of the genome of a host cell, the method comprising:
 (a) introducing into the host cell
 (i) RNA comprising host modifying crRNA (HM-crRNA) and an RNA sequence encoding a host modifying DNA strand with a free end (HM-DNA), wherein the HM-DNA comprises a DNA sequence that is homologous to a sequence in or flanking the target sequence for inserting the HM-DNA into the host cell genome; 
 (ii) a Cas nuclease; and 
 (iii) a reverse transcriptase for producing the HM-DNA from the RNA; and 
   (b) producing the HM-DNA in the host cell using the reverse transcriptase;   wherein the HM-crRNA comprises a nucleotide sequence that hybridizes to the target sequence in the host cell to guide the Cas nuclease to cut the target sequence in the host cell;   wherein the HM-DNA is inserted at a target site comprising the target sequence, and the target sequence is modified.   
     
     
         2 . The method of  claim 1 , wherein the Cas nuclease is a nuclease that cuts in a single DNA strand, but does not make double strand DNA cuts. 
     
     
         3 . The method of  claim 2 , wherein the Cas nuclease is a nickase. 
     
     
         4 . The method of  claim 1 , wherein the Cas is a Cas9. 
     
     
         5 . The method of  claim 4 , wherein the Cas is a  Streptococcus  Cas9. 
     
     
         6 . The method of  claim 5 , wherein the Cas is a  Streptococcus  pyrogenes Cas 9 or a  Streptococcus thermopilus  Cas9. 
     
     
         7 . The method of  claim 4 , wherein the Cas is a  Staphylococcus aureus  Cas 9. 
     
     
         8 . The method of  claim 1 , wherein the HM-crRNA is comprised by a single guide RNA, wherein the single guide RNA comprises a tracrRNA. 
     
     
         9 . The method of  claim 1 , wherein the host cell is a eukaryotic cell. 
     
     
         10 . The method of  claim 9 , wherein the host cell is a human cell, a non-human animal cell, a yeast cell, or a plant cell. 
     
     
         11 . The method of  claim 1 , wherein the host cell is a prokaryotic cell. 
     
     
         12 . The method of  claim 1 , wherein step (a) comprises introducing a nucleic acid vector into the host cell, wherein the vector encodes the HM-crRNA. 
     
     
         13 . The method of  claim 1 , wherein step (a) comprises introducing a nucleic acid vector into the host cell, wherein the vector encodes the RNA sequence for reverse transcription to produce the HM-DNA. 
     
     
         14 . The method of  claim 1 , wherein step (a) comprises introducing a nucleic acid vector into the host cell, wherein the vector encodes the Cas. 
     
     
         15 . The method of  claim 12 , wherein the vector is an AAV or lentiviral vector. 
     
     
         16 . The method of  claim 15 , wherein the host cell is a human cell. 
     
     
         17 . The method of  claim 12 , wherein step (a) further comprises introducing a second nucleic acid vector into the host cell, wherein the second vector encodes the RNA sequence for reverse transcription to produce the HM-DNA. 
     
     
         18 . The method of  claim 1 , wherein the target DNA sequence is an endogenous sequence on a chromosome of the host cell. 
     
     
         19 . The method of  claim 1 , wherein the target DNA sequence is an endogenous sequence on a plasmid of the host cell. 
     
     
         20 . The method of  claim 1 , wherein target site is present in a gene or regulatory element. 
     
     
         21 . The method of  claim 1 , wherein the HM-DNA comprises a regulatory element. 
     
     
         22 . The method of  claim 1 , wherein the method is for medical, dental or ophthalmic use. 
     
     
         23 . A host modifying (HM) CRISPR/Cas system comprising:
 (i) a host modifying crRNA (HM-crRNA) or a nucleic acid sequence encoding the HM-crRNA,   (ii) a Cas nuclease or a nucleic acid sequence encoding the Cas nuclease,   (iii) a reverse transcriptase or a nucleic acid sequence encoding the reverse transcriptase, and   (iv) an RNA or a nucleic acid sequence encoding the RNA, the RNA comprising a sequence that encodes a host modifying DNA strand with a free end (HM-DNA), wherein the reverse transcriptase is capable of producing the HM-DNA from the RNA; and   wherein the nucleic acid sequence encoding the HM-crRNA, the nucleic acid sequence encoding the Cas nuclease, the nucleic acid sequence encoding the reverse transcriptase, and the nucleic acid sequence encoding the RNA are present in one or more nucleic acid vectors.   
     
     
         24 . The HM CRISPR/Cas system of  claim 23 , comprising one or more nucleic acid vectors comprising:
 (i) a nucleic acid sequence encoding the HM-crRNA,   (ii) a nucleic acid sequence encoding the Cas nuclease,   (iii) a nucleic acid sequence encoding the reverse transcriptase, and   (iv) a nucleic acid sequence encoding the RNA comprising the sequence that encodes the HM-DNA.   
     
     
         25 . The HM CRISPR/Cas system of  claim 23 , comprising:
 (i) one or more nucleic acid vectors comprising:
 (a) a nucleic acid sequence encoding the HM-crRNA, 
 (b) a nucleic acid sequence encoding the Cas nuclease, and 
 (c) a nucleic acid sequence encoding the reverse transcriptase; and 
   (ii) the RNA comprising the sequence that encodes the HM-DNA.   
     
     
         26 . The HM CRISPR/Cas system of  claim 23 , comprising:
 (i) the Cas nuclease;   (ii) the reverse transcriptase; and   (iii) one or more nucleic acid vectors comprising:
 (a) a nucleic acid sequence encoding the HM-crRNA, and 
 (b) a nucleic acid sequence encoding the RNA comprising the sequence that encodes the HM-DNA. 
   
     
     
         27 . The HM CRISPR/Cas system of  claim 23 , comprising:
 (i) one or more nucleic acid vectors comprising:
 (a) a nucleic acid sequence encoding the Cas nuclease, and 
 (b) a nucleic acid sequence encoding the reverse transcriptase; and 
   (ii) RNA comprising:
 (c) the sequence that encodes the HM-DNA and 
 (d) the HM-crRNA. 
   
     
     
         28 . A host modifying (HM) CRISPR/Cas system comprising:
 (i) a Cas nuclease;   (ii) a reverse transcriptase;   (iii) a host modifying crRNA (HM-crRNA), and   (iv) a RNA comprising a sequence that encodes a host modifying DNA strand with a free end (HM-DNA), wherein the reverse transcriptase is capable of producing the HM-DNA from the RNA.   
     
     
         29 . A host cell comprising the HM CRISPR/Cas system of  claim 28 .