Methods and compositions for RNA-directed repression of transcription using CRISPR-associated genes

A method for repressing expression of a first target gene in a bacterium or an archaeon, the method comprising introducing into the bacterium or the archaeon:

• (a) a first nucleic acid sequence encoding at least three polypeptides of a Type I CASCADE, wherein the Type I CASCADE is a Type I-B CASCADE, a Type I-C CASCADE, a Type I-E CASCADE, a Type I-F CASCADE, a Type I-A CASCADE or a Type I-D CASCADE;
• and
• (b) a second nucleic acid sequence encoding at least one CRISPR array comprising two or more repeat sequences and at least one spacer sequence that is complementary to a first target sequence from the first target gene, wherein the at least one spacer sequence is increased in length by about 1 to about 100 nucleotides when compared to a normal spacer length, wherein:
• the Type I-B CASCADE comprises a Cas6b polypeptide having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:18-20 or 335-340, a Cas8b (Csh1) polypeptide having at least 95% sequence identity to any one of the sequence of SEQ ID NOs:21, 22, or 1043-1158, a Cas7 (Csh2) polypeptide having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:23, 24, or 1159-1209, and a Cas5 polypeptide having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:25, 26, or 1210-2372,
• the Type I-C CASCADE comprises a Cas5d polypeptide having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:27, 28, or 2373-2973, a Cas8c (Csd1) polypeptide having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:29, 30, or 2974-3847, and a Cas7 (Csd2) polypeptide having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:31, 32, or 3848-4371,
• the Type I-E CASCADE comprises a Cse1 (CasA) polypeptide having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:41, 42, or 5235-6111, a Cse2 (CasB) polypeptide having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:43, 44, or 6112-6823, a Cas7 (CasC) polypeptide having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:45, 46, or 6824-7032, a Cas5 (CasD) polypeptide having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:47, 48, or 7033-7251, and a Cas6e (CasE) polypeptide having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:49, 50, or 7252-7520,
• the Type I-F CASCADE comprises a Cys1 polypeptide having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:51, 52, or 7521-8614, a Cys2 polypeptide having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:53, 54, or 8614-9867, a Cas7 (Cys3) polypeptide having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:55, 56, or 9868-11057, and a Cas6f polypeptide having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:57, 58, or 11058-11528,
• the Type I-A CASCADE comprises a Cas7 (Csa2) polypeptide having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:1-3, a Cas8a1 (Csx13) polypeptide having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:4, or 341-640 or a Cas8a2 (Csx9) polypeptide having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:5, 6, or 641-666, a Cas5 polypeptide having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:7-9, or 667-871, a Csa5 polypeptide having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:872-1042, a Cas6a polypeptide having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:10, 11, or 325-328, a Cas3′ polypeptide having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:12-14, or 329-331, and a Cas3″ polypeptide having no nuclease activity and having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:15-17, or 332-334, said polypeptides having at least 95% sequence identity to one of the sequences of SEQ ID NOs:1-17, 325-334, or 341-1042, and
• the Type I-D CASCADE comprises a Cas10d (Csc3) polypeptide having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:33, 34, or 4372-4678, a Csc2 polypeptide having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:35, 36, or 4679-4985, Csc1 polypeptide having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:37, 38, or 4986-5234, a Cas6d polypeptide having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:39 or 40, thereby repressing expression of the first target gene in the bacterium or archaeon.