Compositions and methods for CRISPR/Cas-based gene editing
The present invention relates to the field of nucleic acid editing, in particular to the field of clustered regularly interspaced short palindromic repeat (CRISPR) technology. Specifically, the present invention relates to Cas effector proteins, fusion proteins comprising such proteins, and nucleic acid molecules encoding them. The present invention also relates to complexes and compositions for nucleic acid editing (e.g., gene or genome editing), which comprise the proteins or fusion proteins of the present invention, or nucleic acid molecules encoding them. The present invention also relates to a method for nucleic acid editing (e.g., gene or genome editing), which uses the proteins or fusion proteins comprising the present invention.
1 . A fusion protein, which comprises a first protein and a second protein or polypeptide; wherein the first protein comprises or consists of a sequence as set forth in any one of SEQ ID NOs: 1, 2, and 3.
2 . The fusion protein according to claim 1 , wherein the second protein or polypeptide is selected from the group consisting of epitope tag, reporter gene sequence, nuclear localization signal (NLS) sequence, targeting moiety, transcription activation domain, transcription repression domain, nuclease domain, and any combination thereof.
3 . The fusion protein according to claim 2 , wherein the fusion protein has an amino acid sequence as set forth in any one of SEQ ID NOs: 28 to 30.
4 . A composition or complex, which comprises:
(i) a first component, which is selected from the group consisting of: the fusion protein according to claim 1 , a nucleotide sequence encoding the fusion protein, and any combination thereof; and
(ii) a second component, which is a nucleotide sequence comprising a guide RNA, or a nucleotide sequence encoding the nucleotide sequence comprising the guide RNA;
wherein, the guide RNA comprises a direct repeat sequence and a guide sequence from the 5′ to 3′ direction, and the guide sequence is capable of hybridizing with a target sequence;
the guide RNA is capable of forming a complex with the fusion protein as described in (i).
5 . The composition or complex according to claim 4 , wherein:
(1) the guide sequence is linked to the 3′ end of the direct repeat sequence;
(2) the guide sequence comprises a complementary sequence of the target sequence; or
(3) the composition or complex does not comprise a trans-activating crRNA (tracrRNA).
6 . The composition or complex according to claim 4 , wherein, when the target sequence is DNA, the target sequence is located at the 3′ end of a protospacer adjacent motif (PAM), and the PAM has a sequence shown as 5′-RYR, wherein R is A or G, and Y is T or C.
7 . The composition or complex according to claim 4 , wherein, the target sequence is a DNA or RNA sequence derived from a prokaryotic cell or a eukaryotic cell; or, the target sequence is a non-naturally occurring DNA or RNA sequence.
8 . The composition or complex according to claim 4 , wherein, the target sequence is present in a cell; or, the target sequence is present in a nucleic acid molecule in vitro.
9 . The composition or complex according to claim 4 , wherein, the fusion protein comprises one or more NLS sequences.