| CPC C12N 9/22 (2013.01) [C12N 15/102 (2013.01); C12N 15/8213 (2013.01); C12N 15/8274 (2013.01); C12N 15/90 (2013.01); C12N 2310/20 (2017.05); C12N 2800/22 (2013.01)] | 12 Claims |
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1. A method for producing a protein having a gain-of-function by targeted alteration of a coding sequence (CDS) in between target sites wherein nontargeted CDS is unaltered in duplex DNA in a cell, comprising
exposing the duplex DNA to at least two site-specific nucleases, wherein a first site-specific nuclease targets the DNA at a first position within the CDS and cleaves the DNA generating a first indel at a first location within the CDS, and wherein a second site-specific nuclease targets the DNA at a second position within the CDS and cleaves the DNA generating a second indel at a second location within the same CDS, wherein the first and second indel are frame shift mutations, resulting in an altered part of the CDS, wherein the altered part of the CDS has a length between 2-15 codons, wherein the CDS before the first indel and after the second indel remain in the same reading frame, wherein the altered part of the CDS starts with the first indel up to and including the second indel, wherein the altered part of the CDS does not comprise a stop codon; and
selecting a cell comprising the targeted alteration,
wherein the CDS comprising the targeted alteration encodes the protein having a gain of function, and
wherein the protein having a gain-of-function is produced with a higher efficiency than with an identical method using a single site-specific nuclease.
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