Myotis lucifugus transposase engineering

A composition comprising (a) a helper enzyme or a nucleic acid encoding the helper enzyme and (b) a targeting element or a nucleic acid encoding the targeting element, wherein:

• the helper enzyme comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:
• 1 and has a non-polar aliphatic amino acid at position 2 of SEQ ID NO:
• 1 or a substitution at a position corresponding thereto,
• one or more amino acid substitutions selected from S8X, C13X, and D416X of SEQ ID NO:
• 1 or a substitution at a position corresponding thereto, and
• one or more amino acid substitutions selected from R333X, K334X, N335X, R336X, Y281X, C282X, G283X, E284X, G285X, K286X, R287X, L288X, L289X, E290X, T291X, I332X, G337X, I338X, and P339X of SEQ ID NO:
• 1 or a substitution at a position corresponding thereto.

A composition comprising a helper enzyme or a nucleic acid encoding the helper enzyme comprising:

• an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:
• 1 and a non-polar aliphatic amino acid at position 2 of SEQ ID NO:
• 1, or a position corresponding thereto, and amino acid substitutions comprising S8X, C13X, R333K, K334A, N335A, R336K, and D416X of SEQ ID NO:
• 1, or a substitution at a position corresponding thereto.

A composition comprising (a) a helper enzyme or a nucleic acid encoding the helper enzyme and (b) a targeting element or a nucleic acid encoding the targeting element, wherein:

• the helper enzyme comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:
• 1 and has a non-polar aliphatic amino acid at position 2 of SEQ ID NO:
• 1 and a substitution at a position corresponding thereto, and amino acid substitutions comprising S8X, C13X, R333K, K334A, N335A, R336K, and D416X of SEQ ID NO:
• 1, or a substitution at a position corresponding thereto.

A composition comprising a helper enzyme or a nucleic acid encoding the helper enzyme comprising:

• an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:
• 1 and a non-polar aliphatic amino acid at position 2 of SEQ ID NO:
• 1, or a position corresponding thereto, and amino acid substitutions comprising S8X, C13X, R333K, N335A, R336K, and D416X of SEQ ID NO:
• 1, or a substitution at a position corresponding thereto.

A composition comprising (a) a helper enzyme or a nucleic acid encoding the helper enzyme and (b) a targeting element or a nucleic acid encoding the targeting element, wherein:

• an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:
• 1 and a non-polar aliphatic amino acid at position 2 of SEQ ID NO:
• 1, or a position corresponding thereto, and amino acid substitutions comprising S8X, C13X, R333K, N335A, R336K, and D416X of SEQ ID NO:
• 1, or a substitution at a position corresponding thereto.

A method of designing a transposase for binding identical target sites upstream and downstream of a target nucleic acid site, comprising:

• a) obtaining the transposase, the transposase being modified to have reduced or ablated inherent nucleic acid binding ability and having at least about 90%, or at least about 93%, at least about 95%, at least about 97%, at least about 98%, at least about 99% identity to the amino acid sequence of SEQ ID NO:
• 1,
• b) fusing or associating the transposase with at least two targeting elements, each targeting element being fused at or immediately after G66 relative to SEQ ID NO:
• 1, optionally wherein the fusing or associating the transposase with at least two targeting elements is by way of a linker,
• wherein the target nucleic acid site is a genomic safe harbor site of or comprising TTAA.