Crispr-cas component systems, methods and compositions for sequence manipulation

A method of selecting one or more prokaryotic cell(s) by introducing one or more mutations in one or more prokaryotic cell(s), the method comprising:

• introducing one or more vectors into the prokaryotic cell(s);
• wherein the one or more vectors drive expression of one or more of:
• a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, a tracr sequence, and an editing template;
• and all of a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, and a tracr sequence, and an editing template are produced in the prokaryotic cell(s);
• introducing the editing template into a target polynucleotide through recombination, wherein the editing template comprises the one or more mutations that abolish CRISPR enzyme cleavage;
• allowing a CRISPR complex to bind to the target polynucleotide to effect cleavage of the target polynucleotide;
• wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to a target sequence within the target polynucleotide, and (2) the tracr mate sequence that is hybridized to a tracr sequence;
• wherein binding of the CRISPR complex to the target polynucleotide induces cell death;
• thereby allowing one or more prokaryotic cell(s) in which one or more mutations have been introduced to be selected.

A method of inducing cell death of one or more prokaryotic cell(s) comprising:

• introducing one or more vectors into the prokaryotic cell(s);
• wherein the one or more vectors drive expression of one or more of:
• a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, and a tracr sequence;
• and all of a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, and a tracr sequence, are produced in the prokaryotic cell(s);
• wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to a target sequence within a target polynucleotide, and (2) the tracr mate sequence that is hybridized to the tracr sequence;
• wherein binding of the CRISPR complex to the target polynucleotide results in Cas9-directed cleavage at a targeted site in the prokaryote(s) and induces cell death.

A method of generating one mutation or multiple mutations in one or more prokaryotic cell(s) by introducing the one mutation or multiple mutations in one or more prokaryotic cell(s) comprising:

• A. generating one mutation in one or more prokaryotic cell(s) by introducing the mutation in one or more prokaryotic cell(s), by a method comprising:
• introducing one or more vectors into the prokaryotic cell(s);
• wherein the one or more vectors drive expression of one or more of:
• a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, a tracr sequence, and an editing template;
• and all of a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, and a tracr sequence, and an editing template are produced in the prokaryotic cell(s);
• allowing a CRISPR complex to bind to a target polynucleotide to effect cleavage of the target polynucleotide whereby the editing template comprising the mutation is introduced through recombination in the one or more prokaryote(s);
• and
• wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to a target sequence within the target polynucleotide, and (2) the tracr mate sequence that is hybridized to a tracr sequence;
• thereby generating one or more prokaryotic cell(s) in which the mutation has been introduced;
• or
• B. generating multiple mutations in one or more prokaryotic cell(s) by introducing the multiple mutations in one or more prokaryotic cell(s), by a method comprising:
• introducing one or more vectors into the prokaryotic cell(s);
• wherein the one or more vectors drive expression of one or more of:
• a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, a tracr sequence, and an editing template;
• and all of a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, and a tracr sequence, and an editing template are produced in the prokaryotic cell(s);
• allowing a CRISPR complex to bind to a target polynucleotide to effect cleavage of the target polynucleotide whereby the editing template comprising the multiple mutations is introduced through recombination in the one or more prokaryote(s);
• and
• wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to a target sequence within the target polynucleotide, and (2) the tracr mate sequence that is hybridized to a tracr sequence;
• thereby generating one or more prokaryotic cell(s) in which multiple mutations have been introduced;
• or
• C. generating multiple mutations in one or more prokaryotic cell(s) by introducing the multiple mutations in one or more prokaryotic cell(s), by a method comprising:
• introducing one or more vectors into the prokaryotic cell(s);
• wherein the one or more vectors drive expression of one or more of:
• a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, a tracr sequence, and an editing template;
• and all of a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, and a tracr sequence, and an editing template are produced in the prokaryotic cell(s);
• allowing a CRISPR complex to bind to a target polynucleotide to effect cleavage of the target polynucleotide whereby the editing template comprising a first mutation is introduced through recombination in the one or more prokaryote(s);
• and
• wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to a target sequence within the target polynucleotide, and (2) the tracr mate sequence that is hybridized to a tracr sequence;
• thereby generating one or more prokaryotic cell(s) in which the first mutation has been introduced;
• introducing one or more vectors into the prokaryotic cell(s) having the first mutation;
• wherein the one or more vectors drive expression of one or more of:
• a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, a tracr sequence, and an editing template;
• and all of a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, and a tracr sequence, and an editing template are produced in the prokaryotic cell(s);
• allowing a CRISPR complex to bind to a target polynucleotide to effect cleavage of the target polynucleotide whereby the editing template comprising a second mutation is introduced through recombination in the one or more prokaryote(s);
• and
• wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to a target sequence within the target polynucleotide, and (2) the tracr mate sequence that is hybridized to a tracr sequence;
• thereby generating one or more prokaryotic cell(s) in which the first and the second mutation has been introduced.

A method for introducing mutation(s) into a prokaryote comprising introducing the mutation(s) by recombineering, and selecting for prokaryote(s) having the mutation(s)

• by introducing one or more vectors into the prokaryotic cell(s);
• wherein the one or more vectors drive expression of one or more of:
• a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, a tracr sequence and an editing oligonucleotide;
• and all of a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, a tracr sequence, and an editing template are produced in the prokaryotic cell(s);
• introducing the editing oligonucleotide comprising the mutation(s) into a target polynucleotide by recombineering,
• allowing a CRISPR complex to bind to the target polynucleotide to effect cleavage of the target polynucleotide, wherein the target sequence is in prokaryote(s) not having the mutation(s) from recombineering;
• and
• wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to a target sequence within the target polynucleotide, and (2) the tracr mate sequence that is hybridized to a tracr sequence;
• wherein binding of the CRISPR complex to the target polynucleotide results in Cas9-directed cleavage at a targeted site in the prokaryote(s) not having the mutation(s) and induces cell death.