Methods for Screening Bacteria, Archaea, Algae, and Yeast Using CRISPR Nucleic Acids

Abstract

This invention relates to the use of CRISPR nucleic acids to screen for essential and non-essential genes and expendable genomic islands in bacteria, archaea, algae and/or yeast, to kill bacteria, archaea, algae and/or yeast, to identify the phenotype of a gene or genes, and/or to screen for reduced genome size and/or a gene deletion in bacteria, archaea, algae and/or yeast.

1 - 30 . (canceled) 
     
     
         31 . A method of selectively killing a specific bacterial subset within a mixed population of bacterial cells, the method comprising:
 introducing into the mixed population of bacterial cells   (a) a heterologous nucleic acid construct comprising a CRISPR array that is functional with an endogenous CRISPR-Cas system present in the specific bacterial subset, and/or   (b) a CRISPR array and a CRISPR-Cas system, wherein the CRISPR array and the CRISPR-Cas system of (b) are introduced on the same or in separate heterologous nucleic acid constructs, in any combination,   wherein the CRISPR array of (a) and (b) comprises (5′ to 3′) a repeat-spacer-repeat sequence or at least one repeat-spacer sequence, and the spacer of the repeat-spacer-repeat sequence or the repeat-spacer sequence comprises a nucleotide sequence that is substantially complementary to a target region in a conserved genetic sequence of the specific bacterial subset that is distinct from other bacterial cells in the mixed population, the target region having at least 10 consecutive nucleotides adjacent to a protospacer-adjacent motif (PAM) recognized by the endogenous CRISPR-Cas system of (a) or the CRISPR-Cas system of (b), thereby selectively killing the specific bacterial subset that comprises the target region.   
     
     
         32 . The method of  claim 31 , wherein the endogenous CRISPR-Cas system of (a) is: (i) a Type I CRISPR-Cas system and the CRISPR array is a Type I CRISPR array, (ii) a Type II CRISPR-Cas system and the CRISPR array is a Type II CRISPR array, (iii) a Type III CRISPR-Cas system and the CRISPR array is a Type III CRISPR array, (iv) a Type IV CRISPR-Cas system and the CRISPR array is a Type IV CRISPR array, or (v) a Type V CRISPR-Cas system and the CRISPR array is a Type V CRISPR array. 
     
     
         33 . The method of  claim 31 , wherein the CRISPR-Cas system of (b) is: (i) a Type I CRISPR-Cas system and the CRISPR array is a Type I CRISPR array, (ii) a Type II CRISPR-Cas system and the CRISPR array is a Type II CRISPR array, (iii) a Type III CRISPR-Cas system and the CRISPR array is a Type III CRISPR array, (iv) a Type IV CRISPR-Cas system and the CRISPR array is a Type IV CRISPR array, or (v) a Type V CRISPR-Cas system and the CRISPR array is a Type V CRISPR array. 
     
     
         34 . The method of  claim 33 , wherein
 (i) the Type I CRISPR-Cas system of (b) comprises (A) at least one Type I Cascade polypeptide and (B) a Cas3 polypeptide, or a Cas3′ polypeptide and a Cas3″ polypeptide, and/or one or more heterologous nucleic acid constructs encoding (A) and/or (B);   (ii) the Type II CRISPR-Cas system comprises a Cas9 polypeptide, and/or a nucleic acid construct encoding the Cas9 polypeptide, optionally wherein the Type II CRISPR-Cas system further comprises a tracrRNA;   (iii) the Type III CRISPR-Cas system comprises at least one Csm complex polypeptide or at least one Cmr complex polypeptide, and/or one or more heterologous nucleic acid constructs encoding the at least one Csm complex polypeptide and/or the at least one Cmr complex polypeptide, and optionally a Cas6 polypeptide or a heterologous nucleic acid construct encoding the Cas6 polypeptide;   (iv) the Type IV CRISPR-Cas system comprises at least one of a Csf4 (dinG) polypeptide, a Csf1 polypeptide, a Csf2 (Cas7) polypeptide and/or a Csf3 (Cas5) polypeptide, and/or one or more heterologous nucleic acid constructs encoding the at least one Csf4 polypeptide, Csf1 polypeptide, Csf2 polypeptide and/or CSf3 polypeptide; and   (v) the Type V CRISPR-Cas system comprises a Cpf1 polypeptide, and/or a heterologous nucleic acid construct encoding the same.   
     
     
         35 . The method of  claim 32 , wherein the repeat-spacer-repeat sequence or the at least one repeat-spacer sequence of: (a) the Type I CRISPR array comprises a repeat that is identical to a repeat from a wild-type Type I CRISPR array, (b) the Type II CRISPR array comprises a repeat that is identical to a repeat from a wild type Type II CRISPR array, (c) the Type III CRISPR array comprises a repeat that is identical to a repeat from a wild type Type III CRISPR array, (d) the Type IV CRISPR array comprises a repeat that is identical to a repeat from a wild type Type IV CRISPR array, or (e) the Type V CRISPR array comprises a repeat that is identical to a repeat from a wild type Type V CRISPR array. 
     
     
         36 . The method of  claim 33 , wherein the repeat-spacer-repeat sequence or the at least one repeat-spacer sequence of: (a) the Type I CRISPR array comprises a repeat that is identical to a repeat from a wild-type Type I CRISPR array, (b) the Type II CRISPR array comprises a repeat that is identical to a repeat from a wild type Type II CRISPR array, (c) the Type III CRISPR array comprises a repeat that is identical to a repeat from a wild type Type III CRISPR array, (d) the Type IV CRISPR array comprises a repeat that is identical to a repeat from a wild type Type IV CRISPR array, or (e) the Type V CRISPR array comprises a repeat that is identical to a repeat from a wild type Type V CRISPR array. 
     
     
         37 . The method of  claim 31 , wherein at least one or more bacterial cells of the mixed population does not comprise the target sequence and, therefore, the at least one or more bacterial cells are not killed upon introduction of the CRISPR array. 
     
     
         38 . The method of  claim 31 , wherein the spacer of the repeat-spacer-repeat sequence or the at least one repeat-spacer sequence comprises a nucleotide sequence that is 100% complementary to the target sequence. 
     
     
         39 . The method of  claim 31 , wherein the target sequence is selected from a gene, open reading frame or a putative open reading frame or an intergenic region, optionally wherein the target sequence is within an essential gene and/or a non-essential gene. 
     
     
         40 . The method of  claim 31 , further comprising introducing into the mixed population of bacterial cells a heterologous nucleic acid construct comprising a trans-encoded CRISPR (tracr) nucleic acid. 
     
     
         41 . The method of  claim 31 , wherein the heterologous nucleic acid construct is comprised in a phage. 
     
     
         42 . A method of identifying in a population of bacterial cells at least one isolate that does not comprise in its genome a conserved target region that is present in the genome of one or more bacterial cells in the population, comprising:
 introducing into the population of bacterial cells   (a) a heterologous nucleic acid construct comprising a CRISPR array that is functional with an endogenous CRISPR-Cas system present in the specific bacterial subset, and/or   (b) a CRISPR array and a CRISPR-Cas system, wherein the CRISPR array and the CRISPR-Cas system of (b) are introduced on the same or in separate heterologous nucleic acid constructs, in any combination,   wherein the CRISPR array comprises (5′ to 3′) a repeat-spacer-repeat sequence or at least one repeat-spacer sequence, and the spacer of the repeat-spacer-repeat sequence or the repeat-spacer sequence comprises a nucleotide sequence that is substantially complementary to a target region in a conserved genetic sequence of the specific bacterial subset that is distinct from other bacterial cells in the mixed population, the target region having at least 10 consecutive nucleotides adjacent to a protospacer-adjacent motif (PAM) recognized by the endogenous CRISPR-Cas system of (a) or the CRISPR-Cas system of (b);   detecting the presence or absence of the conserved target region in the genome of at least one isolate from the population of bacterial cells, and   identifying at least one isolate that does not comprise the conserved target region in its genome.   
     
     
         43 . The method of  claim 42 , wherein the endogenous CRISPR-Cas system of (a) is: (i) a Type I CRISPR-Cas system and the CRISPR array is a Type I CRISPR array, (ii) a Type II CRISPR-Cas system and the CRISPR array is a Type II CRISPR array, (iii) a Type III CRISPR-Cas system and the CRISPR array is a Type III CRISPR array, (iv) a Type IV CRISPR-Cas system and the CRISPR array is a Type IV CRISPR array, or (v) a Type V CRISPR-Cas system and the CRISPR array is a Type V CRISPR array. 
     
     
         44 . The method of  claim 42 , wherein the CRISPR-Cas system of (b) is: (i) a Type I CRISPR-Cas system and the CRISPR array is a Type I CRISPR array, (ii) a Type II CRISPR-Cas system and the CRISPR array is a Type II CRISPR array, (iii) a Type III CRISPR-Cas system and the CRISPR array is a Type III CRISPR array, (iv) a Type IV CRISPR-Cas system and the CRISPR array is a Type IV CRISPR array, or (v) a Type V CRISPR-Cas system and the CRISPR array is a Type V CRISPR array. 
     
     
         45 . The method of  claim 44 , wherein
 (i) the Type I CRISPR-Cas system of (b) comprises (A) at least one Type I Cascade polypeptide and (B) a Cas3 polypeptide, or a Cas3′ polypeptide and a Cas3″ polypeptide, and/or one or more heterologous nucleic acid constructs encoding (A) and/or (B);   (ii) the Type II CRISPR-Cas system comprises a Cas9 polypeptide, and/or a nucleic acid construct encoding the Cas9 polypeptide, optionally wherein the Type II CRISPR-Cas system further comprises a tracrRNA;   (iii) the Type III CRISPR-Cas system comprises at least one Csm complex polypeptide or at least one Cmr complex polypeptide, and/or one or more heterologous nucleic acid constructs encoding the at least one Csm complex polypeptide and/or the at least one Cmr complex polypeptide, and optionally a Cas6 polypeptide or a heterologous nucleic acid construct encoding the Cas6 polypeptide;   (iv) the Type IV CRISPR-Cas system comprises at least one of a Csf4 (dinG) polypeptide, a Csf1 polypeptide, a Csf2 (Cas7) polypeptide and/or a Csf3 (Cas5) polypeptide, and/or one or more heterologous nucleic acid constructs encoding the at least one Csf4 polypeptide, Csf1 polypeptide, Csf2 polypeptide and/or CSf3 polypeptide; and   (v) the Type V CRISPR-Cas system comprises a Cpf1 polypeptide, and/or a heterologous nucleic acid construct encoding the same.   
     
     
         46 . The method of  claim 43 , wherein the repeat-spacer-repeat sequence or the at least one repeat-spacer sequence of: (a) the Type I CRISPR array comprises a repeat that is identical to a repeat from a wild-type Type I CRISPR array, (b) the Type II CRISPR array comprises a repeat that is identical to a repeat from a wild type Type II CRISPR array, (c) the Type III CRISPR array comprises a repeat that is identical to a repeat from a wild type Type III CRISPR array, (d) the Type IV CRISPR array comprises a repeat that is identical to a repeat from a wild type Type IV CRISPR array, or (e) the Type V CRISPR array comprises a repeat that is identical to a repeat from a wild type Type V CRISPR array. 
     
     
         47 . The method of  claim 44 , wherein the repeat-spacer-repeat sequence or the at least one repeat-spacer sequence of: (a) the Type I CRISPR array comprises a repeat that is identical to a repeat from a wild-type Type I CRISPR array, (b) the Type II CRISPR array comprises a repeat that is identical to a repeat from a wild type Type II CRISPR array, (c) the Type III CRISPR array comprises a repeat that is identical to a repeat from a wild type Type III CRISPR array, (d) the Type IV CRISPR array comprises a repeat that is identical to a repeat from a wild type Type IV CRISPR array, or (e) the Type V CRISPR array comprises a repeat that is identical to a repeat from a wild type Type V CRISPR array. 
     
     
         48 . The method of  claim 42 , wherein at least one or more bacterial cells of the mixed population does not comprise the target sequence and, therefore, the at least one or more bacterial cells are not killed upon introduction of the CRISPR array. 
     
     
         49 . The method of  claim 42 , wherein the spacer of the repeat-spacer-repeat sequence or the at least one repeat-spacer sequence comprises a nucleotide sequence that is 100% complementary to the target sequence. 
     
     
         50 . The method of  claim 42 , wherein the target sequence is selected from a gene, open reading frame or a putative open reading frame or an intergenic region, optionally wherein the target sequence is within an essential gene and/or a non-essential gene. 
     
     
         51 . The method of  claim 42 , further comprising introducing into the mixed population of bacterial cells a heterologous nucleic acid construct comprising a trans-encoded CRISPR (tracr) nucleic acid. 
     
     
         52 . The method of  claim 42 , wherein the heterologous nucleic acid construct is comprised in a phage.