Cas9 fusion proteins and related methods

A method of inserting an extraneous sequence into a target region of a genome in a eukaryotic cell, wherein the extraneous sequence for inserting is encoded on a donor DNA molecule comprising a Tn3 core recognition sequence and the target region of a genome comprises a Tn3 core recognition sequence, wherein each Tn3 core recognition sequence is flanked by first and second sgRNA target sequences on opposite strands of the DNA and positioned at equal distances around the Tn3 core recognition sequence, the method comprising:

• introducing into the cell a nucleotide sequence, the nucleotide sequence encoding a fusion protein consisting of the amino acid sequence of SEQ ID NO:
• 1;
• introducing a first oligonucleotide sequence encoding a first single guide RNA (sgRNA) sequence targeting the first sgRNA target sequence and a second oligonucleotide sequence encoding a second sgRNA sequence targeting the second sgRNA target sequence,
• wherein the first and second sgRNA target sequences are spaced to position the 5′ ends of the first and second sgRNAs 22 bp apart, whereby the first and second sgRNAs are on the same helical face of the DNA molecule and the catalytic domains of the hyperactive Tn3 transposon resolvases are positioned to have activity on the Tn3 core recognition sequence;
• introducing the donor DNA molecule encoding the extraneous sequence;
• coexpressing the nucleotide sequence, the first oligonucleotide sequence, the second oligonucleotide sequence, and the donor DNA molecule in the eukaryotic cell to generate a transformed eukaryotic cell;
• and
• culturing the transformed eukaryotic cell to insert the extraneous sequence into the genome of the cultured eukaryotic cell at the site of the target region.

A method of inserting an extraneous sequence into a target region of a genome in a cell, the method comprising:

• introducing into the cell a first nucleotide sequence, the first nucleotide sequence encoding a fusion protein consisting of the amino acid sequence of SEQ ID NO:
• 1;
• introducing a first oligonucleotide sequence encoding a first single guide RNA (sgRNA) sequence, a second oligonucleotide sequence encoding a second sgRNA sequence, and a third oligonucleotide encoding a third sgRNA sequence, wherein:
• the first sgRNA sequence is complementary to the 5′ end of a target region,
• the second sgRNA is complementary to the 3′ end of the target region,
• a protospacer adjacent motif is adjacent and proximal to the 5′ end the target region,
• a protospacer adjacent motif is adjacent and distal to the 3′ end of the target region,
• the distance between the 5′ end of the target region and the 3′ end of the target region is at least 22 bp,
• the target region comprises a sequence complementary to a sequence recognized by the catalytic domain of the recombinase of the fusion protein between the 5′ end of the target region and the 3′ end of the target region,
• the third sgRNA sequence is complementary to a sequence in the genome of the cell that is at least 20 bp from the 3′ end of the target region, wherein the sequence in the genome of the cell that is at least 20 bp from the 3′ end of the target region comprises a protospacer adjacent motif distal to the sgRNA sequence;
• introducing a second nucleotide sequence encoding the extraneous sequence and a recognition site sequence for the fusion protein, wherein:
• the recognition site is proximal to the extraneous sequence, and
• the recognition sequence comprises a sequence complementary to the region of the genome comprising the target region and at least 21 bp from the 3′ end of the target region;
• coexpressing the first nucleotide sequence, the first oligonucleotide sequence, the second oligonucleotide sequence, the third oligonucleotide sequence, and the second nucleotide sequence in the eukaryotic cell to generate a transformed eukaryotic cell;
• and
• culturing the transformed eukaryotic cell to insert the extraneous sequence into the genome of the cultured eukaryotic cell at the site of the target region.