L6_MIIM30011_2024_CRISPR.pdf

 Lecture 6 CRISPR MIIM30011 Dr Mark Davies [email protected] (with special thanks to Prof Hayley Newton)  Learning Objectives At the completion of this lecture students should be able to: explain the role of CRISPR-Cas systems...

 Lecture 6 CRISPR MIIM30011 Dr Mark Davies [email protected] (with special thanks to Prof Hayley Newton)  Learning Objectives At the completion of this lecture students should be able to: explain the role of CRISPR-Cas systems in bacteria describe the key steps of CRISPR-Cas mediated defense discuss the potential application of CRISPR-Cas for genome editing Nature, 24 November 2016 What is CRISPR? Clustered Regularly Interspaced Short Palindromic Repeats – CRISPR -> RNA-guided nuclease system for sequence-specific silencing  Where did it start? 1980s - 1990s: repeat regions in DNA sequences were observed in different microbes by several research groups 1991: Interspaced direct repeats were identified in Mycobacterium tuberculosis complex bacteria 1993: First reported in archaea – Mojica et al End of the 90s: open access to 26 complete microbial genomes and more reports of regularly spaced repeats Short Regular Spaced Repeats (spacer-repeat-spacer-repeat) 2002: Clustered Regularly Interspaced Short Palindromic Repeats - CRISPR Mojica & Rodriguez-Valera The FEBS Journal, 2016 What are CRISPR for? An adaptive immune system for bacteria resistant sensitive An adaptive immune system for bacteria… WHY? Protect prokaryotes from infection by mobile genetic elements Love – Hate relationship. -> Can enhance fitness/adaptation but has a metabolic cost. Enter a balanced state of coexistence BACTERIAL MECHANISMS FOR BLOCKING PHAGE ‘INNATE’ PROCESSES 1. Block phage absorption (receptor modifications) 2. Restriction-modification systems 3. Bacteriophage exclusion (BREX) systems ‘ADAPTIVE’ PROCESSES Robust, heritable immunity Store memory of past infections. CRISPR-Cas Systems CRISPR ASSOCIATED proteins - Cas CRISPR-array (vary) Mohanraju et al., Science 2016 CRISPR-Cas: Class 2 – Type II 3 essential components crRNA (CRISPR array) trans-activating crRNA (complementary to crRNA Repeats) Mali et al., Nature Methods, 2013 Three Stages of CRISPR Immunity 1. ADAPTATION (IMMUNISATION) 2. BIOGENESIS (crRNA MATURATION) 3. INTERFERENCE (IMMUNITY) Hille et al., Cell. 2018 1. Adaptation (immunisation) Mali et al., Nature Methods, 2013 2. Biogenesis (crRNA maturation) Mali et al., Nature Methods, 2013 3. Interference (immunity) Mali et al., Nature Methods, 2013 The PAM sequence Proto-spacer Adjacent Motifs Present on invading viral/plasmid sequence (target DNA) but not part of Cas9: the CRISPR array 2 endonuclease domains Averts ‘auto-immune’ targeting Essential for Cas9 to recognise the sequence and then cleave it Canonical PAM is 5’-NGG-3’ but different Cas9 proteins recognise different PAMs (2-6 base pairs) Hille et al., Cell. 2018 Bacteria – “The Toolmakers” Existence of this system proves that prokaryotic genomes are environmentally tuned They can adapt by Lamarckian inheritance – keeping track of (and inheriting) genomic encounters Points to the hypothesis that there are many more unknown processes yet to be uncovered in the ~30% of function unknown genes that appear in each prokaryotic genome investigated Application of CRISPR Can this system of precise DNA cleavage be harnessed as a genome engineering tool? Class 2, type II system from Streptococcus pyogenes only requires Cas9 and two small RNAs: crRNA and tracrRNA (discovered by Charpentier and Vogel 2011) Chimeric RNA was engineered = sgRNA … single guide RNA able to efficiently direct Cas9 to target DNA (Charpentier, Doudna, Feng Zhang 2012) 2020 Nobel Prize in Chemistry “for the development of a method for genome editing” Genetic scissors: a tool for rewriting the code of life Emmanuelle Charpentier and Jennifer A. Doudna have discovered one of gene technology’s sharpest tools: the CRISPR/Cas9 genetic scissors. Using these, researchers can change the DNA of animals, plants and microorganisms with extremely high precision. This technology has had a revolutionary impact on the life sciences, is contributing to new cancer therapies and may make the dream of curing inherited diseases come true. https://www.nobelprize.org/prizes/chemistry/2020/summary/ Application of CRISPR Tsai and Joung, Nature Collections, CRISPR-CAS9 GENOME EDITING, 2016 Application of CRISPR – Infectious Disease Doerflinger, Cell Microbiol 2017  Application of CRISPR Wang and Qi, Trends in Cell Biology, 2016 Application of CRISPR – Functional genomics deactivated or “dead” Cas9 (dCas9) -> nuclease-deficient variant Cho et al., Int J Mol Sci. 2018  Application of CRISPR Wang and Qi, Trends in Cell Biology, 2016 Application of CRISPR in Host-Pathogen Interaction Research Application of CRISPR: Large Scale Genetic Screens Arrayed screen: reagents are synthesized separately, and constructs are in individual wells. Readout is based on population measurements in individual wells Pooled screen: reagents are synthesized as a pool, readout by next-gen sequencing – comparing abundance of different transgenes in each sample Shalem et al., Nat Rev Genet. 2015 Application of CRISPR: Large Scale Genetic Screens Ma et al., A CRISPR-based screen identified genes essential for West Nile virus-induced cell death. Cell Rep 2015  CRISPR in humans …. ??? https://clinicaltrials.gov/ct2/show/NCT02793856?term=crispr&rank=4 Researchers: removed T-cells from patients with aggressive lung cancer Disabled PD-1 using CRISPR-Cas9 Programmed cell death protein 1 = down-regulates immune response Treatment with PD-1 inhibitors have shown very promising results Re-introduced into patient PD-1 minus cells will attack and defeat cancerous cells https://www.nature.com/articles/d41586-021-01776-4 - Transthyretin amyloidosis (heart disease). ttr gene Human Embryos??? - Extensive ethical considerations (web dive for more debate) http://www.nationalgeographic.com/magazine/2016/08/human-gene-editing-pro-conopinions/ Anti-CRISPR  Anti-CRISPR mechanisms 1. Mutation leading to mismatches and inefficient cleavage. - PAM sequence 2. Recombination in polylysogenic genomes. 3. anti-CRISPR proteins (eg. Acr) - blocks Cas/PAM binding. 4. Phage carrying CRISPR-Cas elements. - Turns CRISPR- against its own host. Hille et al., Cell. 2018 Extra Resources Publications: Throughout lecture Pubmed! Podcast: RadioLab episode: Antibodies Part 1: CRISPR YouTube: https://www.youtube.com/watch?v=MnYppmstxIs 1 problem … new spacers added at the proximal end not the distal end https://www.youtube.com/watch?v=k7X0TBdIk4w Emmanuelle Charpentier https://www.youtube.com/watch?v=TdBAHexVYzc Jennifer Doudna