US 12,442,019 B2
Process for the manipulation of nucleic acids
Amirreza Faridmoayer, Schlieren (CH); Michael Thomas Kowarik, Schlieren (CH); Gerd Martin Lipowsky, Schlieren (CH); and Fabio Serventi, Schlieren (CH)
Assigned to GlaxoSmithKline Biologicals SA, Rixensart (BE)
Filed by GLAXOSMITHKLINE BIOLOGICALS SA, Rixensart (BE)
Filed on Mar. 14, 2024, as Appl. No. 18/605,166.
Application 18/605,166 is a division of application No. 16/635,352, granted, now 11,959,092, previously published as PCT/EP2018/071415, filed on Aug. 7, 2018.
Claims priority of application No. 1712678 (GB), filed on Aug. 7, 2017.
Prior Publication US 2024/0279686 A1, Aug. 22, 2024
Int. Cl. C12N 15/90 (2006.01); C12N 9/10 (2006.01)
CPC C12N 15/902 (2013.01) [C12N 9/1048 (2013.01); C12N 9/1051 (2013.01); C12N 2800/24 (2013.01); C12N 2800/30 (2013.01)] 13 Claims
 
1. A method of removing at least two portions of insert nucleic acid from a genomic polynucleotide in a host cell comprising a host cell genome polynucleotide containing a first recombinantly engineered region, a second recombinantly engineered region, a first recombination site scar and a second recombination site scar,
wherein the first recombination site scar comprises a nucleic acid sequence of the SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and is adjacent to the first recombinantly engineered region in which part of the genomic polynucleotide has been engineered by the addition, deletion, or replacement of a nucleic acid sequence, and the second recombination site scar is adjacent to the second recombinantly engineered region in which part of the genomic polynucleotide has been engineered by the addition, deletion or replacement of nucleic acid sequence;
wherein the first recombinantly engineered region comprises deletion of a waaL gene and the second recombinantly engineered region comprises deletion of at least part of a wca colonic acid cluster and/or deletion of at least part of an rfb cluster,
wherein the first and second recombination site scars have different polynucleotide sequences and the sequence of the second scar is less than 98% identical to SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and wherein the first and second recombination sites are 30-50 base pairs in length and where the first or second recombination site is a FRT site said FRT site is 48 base pairs in length, said method comprising the steps of:
a) preparing the genomic polynucleotide comprising a first insert nucleic acid which is flanked by a pair of first recombination sites in the same orientation which are identical to each other and have a first nucleic acid sequence;
b) exposing the genomic polynucleotide of step a) to a recombinase that recognises the first recombination sites such that the identical recombination sites recombine resulting in the excision of the first insert nucleic acid and one of the first recombination sites;
c) inserting into the genomic polynucleotide of step b) a second insert nucleic acid flanked by a pair of second recombination sites in the same orientation wherein the second recombination sites are identical to each other and have a second nucleic acid sequence which shares no more than 98% sequence identity with the first nucleic acid sequence; and
d) exposing the genomic polynucleotide of step c) to a recombinase that recognises the second recombination sites such that the identical recombination sites recombine resulting in the excision of the second insert nucleic acid and one of the second recombination sites but without the removal of genomic polynucleotide sequence which is not flanked by identical recombination sites.