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Genome Editing, Codon Recoding. �6-Feb-2018 Biophysics 242 George Church

LSRF

NHGRINIGMS

Azco

OppenheimerFoundation

PersonalGenomes.org

Lipper Foundation

v.ht/PHNc

Biophysics 242: Transformative Biotechnologies (R-W-A) Refactored

Jan 23 (1) Exponential, Logistic, Prioritizing global challenges Jan 30 (2) Sequencing & understanding biosphere omes, 3D molecular design Feb 6 (3) Genome Edit/Write (beyond CRISPR), Codon Recoding Feb 13 (4) In situ Sequencing : Mammalian Cell Atlas & BRAIN Initiative Feb 20 (5) Epigenetic programming, signaling pathways, SynEvoDevo Feb 27 (6) Microbiomes: therapeutics, diagnostics, nanopores Mar 6 (7) Synthetic Organs for VUS & Transplantation Mar 20 (8) Aging Reversal Mar 27 (9) Global Warming Apr 3 (10) Germline editing & H. sapiens 2.0

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Reading & Writing Genomes: First semi-synthetic plasmid 1978: $10/b

pBR322 5 genes

NAR 1978Sutcliffe & Church(BR:Bolivar & Rodriguez)

Recombinant DNA vector uses • Human insulin • Human growth hormone • Alpha-interferon • G-CSF • TPA • GM-CSF • Gamma-interferon • IL-2 • Erythropoietin • Hepatitis B vaccine (Amgen, Biogen, Genentech, etc)

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Writing DNA: 1980: $6400 for 10 bases

Gen 1 &2 Oligo Synthesis on chips

5

Phosphoramidite chemistry ~200 sec per base addition300 b max length

Polymerase 10 msec per base>1 Mbase length

Gen 1: $3M per 3 Gb Gen 2: $3K per 3 Gb

Whatcandrivethis?ArchivalInformationstorage

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Genome Project Write. May 2016 Science.

7

2017 Nature Reviews of Genetics, Chari & Church. Beyond editing to writing

large genomes

Add v. Subtract v. Precise edit v. Epigenetic

Genomic locusNuclease

DNA donor (homologous flanks)

Homology Directed Repair

(HDR)

Non-Homologous End Joining (NHEJ)

Random Insertion / Deletion

Ser/TyrIntegrase

Group II intron

Cas9

RecACAGE

Mega-nuclease

λ-RedMAGE

8 Editors: 3 DNA, 2 RNA, 4 protein

ZFN

TALEN

30-90% NHEJ 0% Rough Precise

|--| 1 nm

CRISPR as a technology in eukaryotes

1. Precise editing (HR), not merely cutting / NHEJ.2. Editing normal cells, not just cancer cells (abnormally resistant to DNA cuts).3. Avoiding off-target using computer search of the whole genome.4. Using an 3’ extended sgRNA that works reproducibly in eukaryotic cells.5. Using a RNApol III (e.g. U6) promoter to express the guide RNA.6. Codon-optimized Cas9. A - - - - - - 8-Jun-2012 Jinek … DoudnaB 1 - - - 5 6 3-Jan-2013 Cong … ZhangC 1 2 3 4 5 6 3-Jan-2013 Mali … ChurchD - - - - - - 29-Jan-2013 Cho … KimE - - - 4 5 6 29-Jan-2013 Jinek … Doudna�F - 2 - 4 - - 29-Jan-2013 Hwang … JoungG 1 2 3 4 - 6 2-May-2013 Wang … Jaenisch H - 2 - 4 5 6 4-Apr-2013 Ding … MusunuruI 1 2 3 4 5 6 29-Aug-2013 Ran … Zhang

What’s wrong with CRISPR?

1. Need custom RNA, DNA & protein2. Hard to deliver3. Inefficient once delivered4. Off-target errors5. On target errors 6. Expensive (vs genetic counseling)7. Toxic 8. Large DNA & multiplexing is hard9. Immunity

How was Cas9 an improvement over prior art? ��

Ease of use? Cost? ��

Probably not.�

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Nat Biotechnol. Mar-2013. A library of TAL effector nucleases spanning the human genome. Kim Y … Kim JS “assembled TALEN plasmids for 18,740 protein-coding genes”

HR TALEN vs Cas9

Mali, Yang … Church, Fig 1C Science 3-Jan-2013 Cong … Zhang, Fig 4D

Science 3-Jan-2013

0.37

3.26

8.07HR %

gRNAs

5’ NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAUgcu 3’ :|||||: |||||||| AGUUCAACUAUUGCCUGAUCGGAAUAAAAUU CGAUACGACAAAACUUACCAAGG 5’ A |||| GAA AAAGUGGCACCGA :|||||| G 3’ UUUCGUGGCU

5’ NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGA :|||||: |||| A 3’ GCCUGAUCGGAAUAAAAUU CGAU A GAA

5’ NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGA :|||||: |||| A AGUUCAACUAUUGCCUGAUCGGAAUAAAAUU CGAU A A |||| GAA AAAGUGGCACCGA :|||||| G 3’ UUUCGUGGCU

Cong…Zhang 3-Jan-2013 Fig 2B & S2

Jinek…Doudna 28-Jun-2012 Fig 5B

Mali ... Church 3-Jan-2013 Fig 1A

Computational Jan-2013 Mali …Church, Cong…Zhang SciencePaired nickases Aug-2013 Mali…Church Nat Biotech. Truncated guide RNAs Jan-2014 Fu…Joung Nat Biotech. FokI fusion Apr-2014. Tsai...Joung; Guilinger…Liu Nat Biotech. Brief binding Apr-2015 Davis…Liu. Nat Chem Biol. Weak Cas9-dsDNA Jan-2016 Slaymaker ... Zhang ScienceWeak Cas9-ssDNA Jan-2016 Kleinstiver…Joung Nature.

Still none are reliably SNP specific !!

Genome editing Specificity OFF-target issues

hIPSC HR Jan-2013 Mali …Church. ScienceAsym donor DNA Mar 2016 Richardson … Corn. Nat Biotech BE: C-Deaminase 2009 Yang…Church, 2016 Komor…Liu NaturePost-HDR NHEJ May-2016 Pacquet ... Tessier-Lavigne. Nature Apoptosis toxicity 2016 submitted Byrne … Church

3 Alternatives to ds-breaks: Ser/Tyr Integrase/recombinase AAV editing λ-Red recombinase

Beyond CRISPR: ON-target issues (& large chunks)

17 Harris Wang et al Nature 2009

5-fold improvement in 3 days

AND improve growth rates

Accelerated Evolution 23K RBS combinations per gene Lycopene (hydrocarbon): 20 genes up, 4 down, 2 new

8 Optimizations for ss allele replacement

Oligo length 10x

Phosphorothioate 3x

ΔG > –13 kcal/mol 30x

[oligo] = 0.05-50uM

Wang, et al. Nature 2009 Isaacs et al Science 2011 Ellis et al PNAS 2001

Red-β >10,000x MutS 100x

Lagging strand: 30x Co-selection 4x

Genome-scale precise editing�4 Million bp Genome Recoding: 63 & 57 codon types

Science 2016 Ostrov, et al Nature 2015 Mandell et al Science 2013 Lajoie et al2. Genetic &

Metabolic Isolation

3. Multi-Virus resistance

1. Non-standard amino acids (NSAA)321 & 62,214 codons

Codon types impacting all viruses

Ostrov et al Science 2016

UAGStop

+ rare ArgAGAAGG

UUA LeuUUG orAGC SerAGU

ß 4/6 viruses zero yield from 1012 titer

Genetically encoded unnatural amino acids Liu & Schultz 2010 Ann Rev Biochem.

Non-standard Amino Acids

Ketone 9 Orthogonal chem

Azobenzyl 14 Photoisomers

Coumarinyl 13 Redox

Cytosinyl 8 Peptide-NA

Dansyl 17 Fluorescent

Biotinyl-Lys 19 Binds Avidin

Side Chain Key AAAA #atoms(non-H) Features

Clinical tests of non-standard AA “orthogonal” chemistry

PEG-pAcPhe-hGHAmbrx,Cho,Schultzetal.higherserumstability

+hydrazideKetone hydrazone

24 24

Improving a Natural Enzyme Activity through Incorporation of Unnatural Amino Acids -- Ugwumba et al 2010 JACS

8-11-fold improvement … in contrast to … screening hundreds of thousands of mutants with natural amino acids.

Ecologicallyisolatedcells.(metabolically&genetically)

Failure modes: •  Near-cognate suppression •  tRNA anticodon mutations •  UAG reversion

Solutions: •  Exclude canonical amino acids •  Require compensatory mutations •  Reduce spontaneous mutation freq.

•  mutS-- 2x10-8 •  mutS+ 10-10

Starting with 120 known crystal structures

Mandell et al Nature 2015

MarcLajoieDanMandellRyoTakeuchiBarryStoddard

TyrS WT only

TyrS WT + TyrS-des7 design (cyan)

X-ray with electron density contoured at 1.0σ

X-ray with design(cyan)

NSAAs in 5 essential genes.�

Escape frequencies

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J. Responsible Innovation. 2017 Harnessing gene drive. Min, Smidler, Najjar, Esvelt

Nature. 2016 Turning point Nature. 2016 Gene editing can drive science to openness. Sci Am. 2017 Unnatural Responsibilities. Science. 2017 Precaution: Open gene drive research. PLoS Biol. 2017 Conservation demands safe gene drive.

EsveltetaleLife

Gene Drives for the Alteration of Wild Populations

Reversal & Local Daisy Drives

Noble et al. Biorxiv 2016

Problems. 1A: Please list your help sources.1B: Quantitate approaches to climate change 1C: Deceleration of ng-scale starshot? What to take? Build communication (3D printer) on arrival? 2A: Fetch Y from Genbank. Code to find codons. 2B: Methods to understand Omes.2C: Odds of correct segregation of all human chromosomes if movement is random? 3A: Recode one (or 53) Y genes. Leu UUR to CUX3B: Consequences of such recoding? How to test?

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