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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
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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.
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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”
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?