Recombinant type I CRISPR-Cas system
This invention relates to recombinant Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) arrays and recombinant nucleic acid constructs encoding Type I-E CASCADE complexes as well as plasmids, retroviruses and bacteriophage comprising the same.
1. A recombinant nucleic acid construct comprising a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) array comprising two or more repeat sequences and one or more spacer sequence(s), wherein each of the one or more spacer sequences is linked at its 5′ end and at its 3′ end to a repeat sequence, and each of the one or more spacer sequences is complementary to one or more target sequences in a target DNA of a target organism, wherein the target DNA is located immediately adjacent (3′) to a protospacer adjacent motif (PAM), wherein the two or more repeat sequences comprise 20 to 28 consecutive nucleotides of any one of the nucleotide sequences of SEQ ID NOs:1, 10, 19, 28, 37, 42, 51, or 60.
2. The recombinant nucleic acid construct of claim 1 , wherein the PAM comprises a nucleotide sequence of 5′-NAA-,3′ 5′-AAA-3′ or 5′-AA-3′ that is immediately adjacent to and 5′ of the target sequence.
3. The recombinant nucleic acid construct of claim 1 , wherein the spacer sequence is about 80 to 100% complementary to the target sequence.
4. The recombinant nucleic acid construct of claim 1 , wherein the one or more spacer sequence(s) each have a length of about 25 nucleotides to about 40 nucleotides.
5. The recombinant nucleic acid construct of claim 1 , wherein the one or more spacer sequence(s) each comprise a 5′ region and a 3′ region, wherein the 5′ region comprises a seed sequence and the 3′ region comprises a remaining portion of the one or more spacer sequence(s).
6. The recombinant nucleic acid construct of claim 5 , wherein the seed sequence comprises the first 8 nucleotides of the 5′ end of each of the one or more spacer sequence(s), and is fully complementary to the target sequence, and the remaining portion of the one or more spacer sequence(s) is at least 80% complementary to the target sequence.
7. The recombinant nucleic acid construct of claim 1 , wherein the target sequence is located in a gene, optionally in the sense or coding strand or in the antisense or non-coding strand.
8. The recombinant nucleic acid construct of claim 7 , wherein the target sequence is located in an intragenic region of the gene, optionally located in the sense or coding strand or in the antisense or non-coding strand.
9. The recombinant nucleic acid construct of claim 1 , wherein the target sequence is located in an intergenic region.
10. The recombinant nucleic acid construct of claim 7 , wherein the target sequence is located on a chromosome, a mobile element, an extrachromosomal nucleic acid or a plasmid.
11. The recombinant nucleic acid construct of claim 7 , wherein the gene encodes a transcription factor or a promoter.
12. The recombinant nucleic acid construct of claim 7 , wherein the gene encodes non-coding RNA (e.g., miRNA, siRNA, piwi-interacting RNA (piRNA) and long non-coding RNA (lncRNA).
13. The recombinant nucleic acid construct of claim 1 , wherein the target organism is a prokaryote or a eukaryote.
14. A recombinant cell comprising the recombinant nucleic acid construct of claim 1 .
15. A vector encoding the recombinant nucleic acid of claim 1 .
16. The vector of claim 15 , further comprising a recombinant nucleic acid encoding a Type I-E CRISPR associated complex for antiviral defense complex (Cascade complex) comprising a Cse1 polypeptide, a Cse2 polypeptide, a Cas7 polypeptide, a Cas5 polypeptide and a Cas6 polypeptide.
17. The vector of claim 16 , wherein the Cse1 polypeptide is encoded by the nucleotide sequence of SEQ ID NO:82, the Cse2 polypeptide is encoded by the nucleotide sequence of SEQ ID NO:83, the Cas7 polypeptide is encoded by the nucleotide sequence of SEQ ID NO:84, the Cas5 polypeptide is encoded by the nucleotide sequence of SEQ ID NO:85, and the Cas6 polypeptide is encoded by the nucleotide sequence of SEQ 11) NO:86.
18. The vector of claim 16 , further comprising a polynucleotide encoding a Cas3 polypeptide, wherein the polynucleotide encoding the Cas3 comprises the nucleotide sequence of SEQ ID NO:87 or wherein the polynucleotide encoding the Cas3 encodes the amino acid sequence of SEQ ID NO:119.
19. The vector of claim 15 , wherein the vector is a plasmid, bacteriophage, and/or retrovirus.