DNA PROTEASE CUTTING AGENT BASED ON CAS9 PROTEIN FROM PASTEURELLA PNEUMOTROPICA BACTERIA Russian patent published in 2020 - IPC C12Q1/68 

FIELD: biotechnology.

SUBSTANCE: invention relates to the biotechnology. Described is the use of a protein capable of forming a double-strand break in a DNA sequence directly adjacent to sequence 5'-NNNN(A/G)T-3' containing the amino acid sequence SEQ ID NO: 1 or containing amino acid sequence, which is at least 95 % identical to the amino acid sequence SEQ ID NO: 1 and has differences compared to SEQ ID NO: 1 only in non-conservative amino acid residues, to form double-strand break in DNA molecule located immediately before nucleotide sequence 5'-NNNN(A/G)T-3' in said DNA molecule. In addition, described is a method of forming a double-strand break in a sequence of genomic DNA of a unicellular or multicellular organism directly adjacent to sequence 5'-NNNN(A/G)T-3'. Introducing at least one cell of said organism an effective amount of a) a protein comprising the amino acid sequence SEQ ID NO: 1 and capable of forming a double-strand break in the DNA sequence directly adjacent to sequence 5'-NNNN(A/G)T-3', or nucleic acid coding said protein, and b) a RNA guide containing a duplex-forming sequence with a nucleotide sequence of a genomic DNA portion of the organism immediately adjacent to nucleotide sequence 5'-NNNN(A/G)T-3', and interacts with said protein after formation of duplex, or DNA sequence coding said RNA guide. Interaction of said protein with the RNA guide and 5'-NNNN(A/G)T-3' nucleotide sequence leads to formation of double-strand break in a sequence of genomic DNA directly adjacent to sequence 5'-NNNN(A/G)T-3'.

EFFECT: thereby achieving higher universality of available CRISPR-Cas9 systems, which will make it possible to use Cas9 nucleases from different organisms for cutting genome or plasmid DNA in more specific sites and under different conditions.

5 cl, 13 dwg, 2 tbl, 4 ex