	US 12,221,617 B2
	SCRaMbLE of heterozygous diploid yeast
Michael Shen, New York, NY (US); Jef D. Boeke, New York, NY (US); and Yi Wu, Tianjin (CN)
Assigned to New York University, New York, NY (US)
Appl. No. 16/315,846
Filed by New York University, New York, NY (US)
PCT Filed Jul. 7, 2017, PCT No. PCT/US2017/041194 § 371(c)(1), (2) Date Jan. 7, 2019, PCT Pub. No. WO2018/009863, PCT Pub. Date Jan. 11, 2018.
Claims priority of provisional application 62/359,403, filed on Jul. 7, 2016.
Prior Publication US 2019/0300910 A1, Oct. 3, 2019
Int. Cl. C12N 15/90 (2006.01); C12N 1/18 (2006.01); C12N 3/00 (2006.01); C12N 15/10 (2006.01); C12N 15/52 (2006.01); C12N 15/81 (2006.01)

CPC C12N 15/905 (2013.01) [C12N 1/18 (2013.01); C12N 3/00 (2013.01); C12N 15/102 (2013.01); C12N 15/52 (2013.01); C12N 15/81 (2013.01); C12N 2800/30 (2013.01)]

15 Claims

1. A method for making a diploid yeast having heterozygosity for at least one chromosome, the method comprising:

i) mating haploid yeast having at least a first modified chromosome comprising a synthetic chromosome capable of recombination-site-mediated evolution (a SCRaMbLE-ready modification) wherein the SCRaMbLE-ready modification comprises introduced site-specific recombinase recognition sites that can be recognized by a recombinase, with a haploid yeast devoid of the SCRaMbLE-ready modification to obtain diploid SCRaMbLE-ready yeast;

ii) subsequent to the mating, using the recombinase to recombine the first modified chromosome to obtain heterozygous diploid yeast comprising at least one recombined (SCRaMbLEd) chromosome and a homologous non-SCRaMbLEd chromosome, and

iii) identifying heterozygous diploid yeast that comprise the at least one SCRaMbLEd chromosome and which have a changed phenotype that is different from the phenotype of the diploid SCRaMbLE-ready yeast.