genome editing P A W A N K . D H A R School of Biotechnology - PowerPoint PPT Presentation

genome editing P A W A N K . D H A R School of Biotechnology Jawaharlal Nehru University A QUICK TECHNICAL WALK THROUGH New Delhi 1 of 18 https://upload.wikimedia.org/wikipedia/commons/8/8b/Ácido_desoxirribonucleico_%28DNA%29.png

Restriction Enzyme DNA 5’… CCC GGG… 3’ 3’… GGG CCC… 5’ B LU N T E N D R E S T R I C T I O N S I T E Cutting the DNA 2 of 18

S T I C K Y E N D R E S T R I C T I O N S I T E GAATTC CTTAAG Cutting the DNA 3 of 18

Three possibilities s l i c i n g D N A l e a d s t o s e v e r a l p o s s i b i l i t i e s DNA TRANSGENE ADDITION GENE DISRUPTION GENE CORRECTION (adding a new gene) (homologous recombination) 4 of 18

Challenge ONE : To achieve targeted integration COLLATERAL BULL’S EYE DAMAGE (LOW (MORE OCCURENCE) FREQUENT) 5 of 18

CHALLENGE ONE : Targeted integration n 4 Frequency of cutting the DNA is 6 4 EcoRI: 6 base recognition sequence = 4096 E. Coli genome length ~ 4.6 million bp assuming uniform distribution 4, 600, 000/ 4096 of restriction sites 1123 6 of 18

Challenge TWO : To make cellular uptake more efficient 7 of 18

TOOLS FOR GENOME SURGERY TRANSCRIPTION ACTIVATOR ZINC FINGER NUCLEASES LIKE EFFECTOR NUCLEASES ZFNs TALENs D N A B I N D I N G P R O T E I N 8 of 18 E N D O N U C L E A S E C A T A L Y T I C D O M A I N

Latest tool in genome editing CRISPR CAS9 Cas9 guide RNA 9 of 18

detailed description CRISPR CAS9 Csa9 guide RNA DNA non seed seed NGG 8 nt 12 nt 3 nt Protospacer PAM 20 nt Protospacer Adjacent Motif 10 of 18

Applications of Genome Engineering Hsu et al 2014. Cell 157, 1262-78 11 of 18

July 2020 control CRISPR 1 2 CRISPR-Cas Φ a h y p e r c o m p a c t g e n o m e e d i t o r Cas9 : 163 kDa Cas Φ : 70 KDa Deleted TDO gene Success rate > 90% The Marine Biological Laboratory, MA, USA 2020 Science Jul 17;369:333-337 Karen Crawford et al. Highly Efficient Knockout of a Squid 12 of 18 Pigmentation Gene. Current Biology, July 30, 2020

A CRISPR approach to treating sickle cell disease 3 13 of 18 Wu et al Nat Med 2019: 25, 776-83

Repurposing bacterial toxins for DNA surgery 4 CRISPR FREE GENOME EDITING DddA : double-stranded DNA cytidine deaminase 14 of 18 Image source: https://www.sciencemag.org/news/2020/07/new-method-edit-cell-s-powerhouse-dna-could-help-study-variety-genetic-diseases

Human genome editing 5 Expert Advisory Committee developing Global Standards for Governance and Oversight of Human Genome Editing 15 of 18

Used CRISPR-Cas9 to reduce the transmission efficiency of malaria parasites in mosquitoes Prof. Shailja Singh Jawaharlal Nehru University Singh et al. Proc Natl Acad Sci U S A. 2019: 116(35): 17498 – 17508 16 of 18

Protein RNA NOT Dhar et al 2009 coding coding Joshi et al 2013 coding Raj et al 2015 Shidhi et al 2015 Joshi et al 2016 Varughese et al 2017 Making functional proteins from the non expressing sequences of the genome tREP: tRNA encoded peptide antimalarial Anti breast cancer Anti Alzheimers Anti bacterial Anti fungal Anti leishmania FORESIGHT BIOTECH Pvt. Ltd. (patent filed, publication coming very soon) Jawaharlal Nehru University, New Delhi 17 of 18

Identified novel Cas like molecules from Intergenic sequences of model organisms patent : under process Anti Cas proteins Thank you dharlab.net 18 of 18