US 12,116,570 B2
Directed evolution of enzymes by plasmid tagging in droplets
Mark Stamatios Kokoris, Bothell, WA (US); Michael Kovarik, Seattle, WA (US); Marc Prindle, Seattle, WA (US); Salka Keller, Shoreline, WA (US); Robert Busam, Seattle, WA (US); and Miranda Lahman, Seattle, WA (US)
Assigned to Roche Sequencing Solutions, Inc., Pleasanton, CA (US)
Appl. No. 16/337,282
Filed by Roche Sequencing Solutions, Inc., Pleasanton, CA (US)
PCT Filed Sep. 28, 2017, PCT No. PCT/US2017/054021
§ 371(c)(1), (2) Date Mar. 27, 2019,
PCT Pub. No. WO2018/064338, PCT Pub. Date Apr. 5, 2018.
Claims priority of provisional application 62/412,693, filed on Oct. 25, 2016.
Claims priority of provisional application 62/401,780, filed on Sep. 29, 2016.
Prior Publication US 2020/0032247 A1, Jan. 30, 2020
Int. Cl. C12N 15/10 (2006.01); C12N 1/20 (2006.01); C12N 9/00 (2006.01); C12N 9/12 (2006.01); C12N 15/74 (2006.01); C40B 20/04 (2006.01); C40B 40/08 (2006.01)
CPC C12N 15/1058 (2013.01) [C12N 1/20 (2013.01); C12N 9/1252 (2013.01); C12N 9/93 (2013.01); C12N 15/74 (2013.01); C40B 20/04 (2013.01); C40B 40/08 (2013.01); C12Y 207/07007 (2013.01)] 5 Claims
 
1. A method for the selection of an enzyme capable of modifying a nucleic acid, wherein the method is not dependent on the complete replication of the enzyme-encoding gene, the method comprising the steps of:
(a) providing a parent nucleic acid encoding an enzyme of interest, wherein the parent nucleic acid provides the gene sequence of the enzyme of interest, wherein the enzyme of interest is a DPO4 DNA polymerase, and wherein the parent nucleic acid is provided in a host cell;
(b) providing conditions to induce the host cell to produce the enzyme of interest such that the host cell comprises the parent nucleic acid together with the enzyme of interest;
(c) following production of the enzyme of interest by the host cell, compartmentalizing the host cell, wherein the step of compartmentalizing the host cell comprises forming a water in oil emulsion droplet, wherein the water in oil emulsion droplet comprises the host cell together with a lysozyme, a nickase endonuclease, and XNTPs, wherein the XNTPs each comprise a selectively cleavable bond and a tether that is attached at positions that allow for controlled expansion of the XNTP by intra-XNTP cleavage of the selectively cleavable bond; and wherein the lysozyme is capable of lysing the host cell to release the parent nucleic acid and the enzyme of interest;
(d) providing conditions to cleave at least one strand of the parent nucleic acid to provide at least one free 3′ end and at least one free 5′ end;
(e) providing conditions such that the at least one free 3′ end or the at least one free 5′ end of the parent nucleic acid is modified by the enzyme of interest to yield a modified parent nucleic acid, wherein the modified parent nucleic acid comprises XNTPs and a molecular tag; and
(f) isolating the modified parent nucleic acid.