	US 11,746,347 B2
	Simultaneous multiplex genome editing in yeast
Miles Gander, Boulder, CO (US); Tian Tian, Boulder, CO (US); Skylar Stefani, Boulder, CO (US); and Patrick Westfall, Boulder, CO (US)
Assigned to Inscripta, Inc., Pleasanton, CA (US)
Filed by Inscripta, Inc., Boulder, CO (US)
Filed on Feb. 24, 2022, as Appl. No. 17/679,203.
Application 17/679,203 is a continuation of application No. 17/463,956, filed on Sep. 1, 2021, granted, now 11,274,296.
Application 17/463,956 is a continuation of application No. 17/230,241, filed on Apr. 14, 2021, granted, now 11,136,572, issued on Oct. 5, 2021.
Application 17/230,241 is a continuation of application No. 17/061,143, filed on Oct. 1, 2020, granted, now 11,001,831, issued on May 11, 2021.
Application 17/061,143 is a continuation in part of application No. 16/827,639, filed on Mar. 23, 2020, granted, now 10,815,467, issued on Oct. 27, 2020.
Claims priority of provisional application 62/960,291, filed on Jan. 13, 2020.
Claims priority of provisional application 62/871,879, filed on Jul. 9, 2019.
Claims priority of provisional application 62/823,136, filed on Mar. 25, 2019.
Prior Publication US 2023/0012154 A1, Jan. 12, 2023
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 15/10 (2006.01)

CPC C12N 15/1037 (2013.01)

24 Claims

1. A method for RNA-directed nuclease editing comprising the steps of:

designing and synthesizing a first linear vector backbone library comprising a coding sequence for a nuclease, a coding sequence for a first portion of a first antibiotic resistance gene fused to an N-terminus of a first intein, and first homology regions for inserting a first library of editing cassettes;

designing and synthesizing a second linear vector backbone library comprising a coding sequence for a nuclease, a coding sequence for a second portion of a first antibiotic resistance gene fused to an C-terminus of the first intein, and second homology regions for inserting a second library of editing cassettes;

designing and synthesizing the first library of editing cassettes, wherein each editing cassette comprises (i) a sequencing encoding a gRNA (ii) and a donor DNA, and wherein homology exists between the first library of editing cassettes and the first linear vector library;

designing and synthesizing the second library of editing cassettes, wherein each editing cassette comprises (i) a sequence encoding a gRNA and (ii) a donor DNA, and wherein homology exists between the second library of editing cassettes and the second linear vector library;

transforming cells with the first and second linear vector backbone libraries and first and second libraries of editing cassettes;

selecting for transformed cells by selecting for antibiotic resistance to the first antibiotic; and

providing conditions for RNA-directed nuclease editing in the cells to produce first edited cells.