Leslie MitchellNew York UniversityLangone Health

www.synthe)cyeast.org

Imagecredit:SarahRichardson

Lessons learned from Sc2.0, the Synthetic Yeast Genome Project

@syngenome

•  Design&buildanen)relysynthe)ceukaryo)cgenometodirectgrowthofS.cerevisiae

•  Improvegene)cflexibilityandgenomicstabilitywhilemaintaininghighfitness

Sc2.0–TheSynthe.cYeastGenomeProject

Sc2.0–designfeatures

Delete Re-locate Add Re-code

Repeats(transposons,LTRs,Sub-telomerers)

Introns

tRNAgenesmovedtoanewchromosome

loxPsymsitesin3’UTRsandotherlandmarks;‘SCRaMbLE’

Watermarksequencesforscreening(PCRTags)

‘Stopswaps’(TAGtoTAA)

•  Improvedgenomestability •  Induciblegene)cflexibility

•  Iden)fica)on•  Biosecurity•  Newprotein

chemistry

Meandistancebetweenclustersofedits=400bp

Sc2.0designergenomesequence

‘Sc1.0’ ‘Sc2.0’

Richardsonetal.2017

BiostudioDesignSo_ware–JoelBader,SarahRichardson,GiovanniStracquadanio,KunYang

Thebuildchallenge:HowdoesonephysicallyconstructSc2.0DNAfor

deliverytoyeastcells?

Minichunks:3kb

Megachunks:30-60kb

Chunks:10kb

Oligos:70bp

Buildingblocks:750bp

SourcesofDNA:SynthesiscompaniesUndergrads!(B-A-G)

Richardsonetal.2017

Build30-60kb‘megachunks’invitro ExploityeastHRfordeliverybyreplacement

Thedeliverychallenge:Howdoesoneassemblechromosomesinyeast?

Thedebuggingchallenge:

“PooledPCRTagmapping(PoPM)”

Wuetal.Science2017

Howdoesonerapidlymapandcorrectbugs?

Xieetal.Science2017

Mul)plexedCRISPR-Cas9edi)ng

Sc2.0Interna)onalConsor)um

Richardsonetal.2017

Richardsonetal.Science2017,Mitchelletal.Science2017

515kbsynthe)cDNA(6%ofgenome)

75kbdeleted12kbre-coded7kbadded

94kbchanged

Endoreduplica)onintercross

Thefinalchallenge:HowdoesoneconsolidateSc2.0chromosomes?

Genomewri)ngchallenges:

•  Whatareyougoingtobuild…andWHY?•  Agrandchallenge…oragrantchallenge?•  It’sgoingtobeabigjob,whowillpar)cipate?•  Whatisthedefini)onofsuccess?

Idea Design SourceDNA Build

Measure&DebugDeliver

JefBoekeJingchuanLuoYuZhao

JoelBaderS.ChandrasegaranG.StracquadanioKunYangSarahRichardsonHeloiseMullerVisuAnnaluruBAGstudentsKarenZelleriGEM2011,2012

SakkiePretoriusDanJohnsonNatalieCurachTomWilliams

JunbiaoDaiWeiminZhang

PatrickCai

YJYuanYiWuZexiongXieMengZhaoBinJia

MaMhewChangAddisonWong

TomEllisBenBlount

HuamingYangYueShen

Sc2.0Thanks:

RomainKoszulGuillaumeMercy

MichaelShenJessicaDymondAndrewMar)n

YasunoriAizawaHitoyoshiYamahitaHikaruKurasawa