Genome Editing II CRISPR/Cas9 PDF

Genome editing II CRISPR/Cas9  Geography of CRISPR discovery Cell. 2016 Jan 14;164(1-2):18-28. doi: 10.1016/j.cell.2015.12.041. © 2016 Elseiver Inc. Learning objectives Define the importance of the CRISPR/Cas system in bacterial 1. immunity 2. systems Identify the components...

Genome editing II CRISPR/Cas9  Geography of CRISPR discovery Cell. 2016 Jan 14;164(1-2):18-28. doi: 10.1016/j.cell.2015.12.041. © 2016 Elseiver Inc. Learning objectives Define the importance of the CRISPR/Cas system in bacterial 1. immunity 2. systems Identify the components of the CRISPR/Cas9 and Cpf1 Describe in detail the mechanism of action of CRISPR/Cas9 in 3. genome editing Discuss potential applications of CRISPR/Cas9 to biology and 4. biotechnology Emmanuelle Charpentier © 2017 CeMM 1. Bacterial immunity CRISPR/Cas system - bacterial immunity Clustered regularly interspaced short palindromic repeats (CRISPR) A bacterial immunity system to protect against phages Elements of the phage DNA become part of the CRISPR array or locus These small fragments of DNA can be replicated to synthesize crRNA crRNA targets effector proteins to foreign Trends Genet. 2014 Mar;30(3):111-8. doi: 10.1016/j.tig.2014.01.003 © 2014 Elseiver Inc. DNA in the cell  CRISPR system is multifaceted Different types of CRISPR system exist Type I-VI CRISPRs are constructed of different components that vary depending on mechanism Mechanisms of all types are not fully understood Type II CRISPR systems will be the system of interest in this course Cell. 2016 Jan 14;164(1-2):29-44. doi: 10.1016/j.cell.2015.12.035. © 2016 Elseiver  Structure of 2. CRISPR/Cas9 System  Type II systems used in mammalian gene editing Type II CRISPR systems use Cas9 as the effector nuclease This system requires two components: tracRNA and crRNA These target the Cas9 nuclease to the locus where a DSB is induced Cell. 2016 Jan 14;164(1-2):18-28. doi: 10.1016/j.cell.2015.12.041. © 2016 Elseiver Inc. Mechanisms of action vary by system The previously mentioned components of tracRNA and crRNA are required for Cas9 DNA cleavage These target the Cas9 nuclease to the locus where a DSB is induced Together they form what is referred to as the gRNA A specific sequence motif, protospacer adjacent motif (PAM), indicates where Cas9 DNA cleavage will occur Cell. 2014 Jun 5;157(6):1262-78. doi: 10.1016/j.cell.2014.05.010. © 2014 Elseiver Inc. In bacteria this leads to the degradation of foreign DNA CRISPR/Cas9  Cpf1 as an alternative to Cas9 Cpf1 is a type V CRISPR system nuclease Cpf1 crRNAs are processed as is and do not require a tracRNA PAM sequence of Cpf1 is T rich Cpf1 contains one DNA cleavage domain in common with Cas9 dsDNA breaks are staggered double stranded Cell. 2015 Oct 22;163:759-771. doi: 10.1016/j.cell.2015.09.038. © 2015 Elseiver 3. Mechanism of DNA cleavage Business end of Cas9 Cas9 contains two domains which possess nuclease activity These are the RuvC and HNH domains Each of these is responsible for cutting one strand of DNA The RuvC domain cuts the strand at a position adjacent to the PAM Cell. 2014 Jun 5;157(6):1262-78. doi: 10.1016/j.cell.2014.05.010. © 2014 Elseiver Inc. While the HNH domain cuts on the strand to which the gRNA is directly hybridised CRISPR/Cas9 in eukaryote genome editing Business end of Cpf1 Recent data supports Cpf1 having a single catalytic cleavage site for both DNA strands This cleavage site is in the RuvC domain The Nuc domain contributes to DNA binding but not to the catalysis of cleavage The exact mechanism of cleavage Molcular Cell. 2017 Oct 5;157(6):15-25. doi: 10.1016/j.molcell.2017.09.007. © 2017 Elseiver Inc. is still unclear Cpf1 and Cas9 differences in structure Domains Wedge (WED) RuvC REC HNH (RECI/II) His-Asn-His Recognition domain domain PAM Bridging Spring 8 Research Frontiers. 2016. © 2016 interacting helix (BH) (PI) Cpf1 crystal structure Nature. 2017 Jun 22; 546:559-563. doi: 10.1038/nature22398. © 2016 Macmillian Publishers  CRISPR 4. Applications Possible applications of CRISPR/Cas A- nuclease nicking activity B- double nicking with nuclease C- single plasmid containing all components D- Injection of Cas9 protein into embryos E- viral application of all CRISPR/Cas9 components F- High throughput application of CRISPR/Cas9 for genetic libraries G- Transcriptional or epigenetic control of gene expression H- Labelling of specific gene loci I- Induction of gene regulation based on environmental conditions Cell. 2014 Jun 5;157(6):1262-78. doi: 10.1016/j.cell.2014.05.010. © 2014 Elseiver Inc. Feng Zhang © 2017 MIT CRISPR/Cas9 in eukaryote genome editing Mammalian genome editing Cell. 2014 Jun 5;157(6):1262-78. doi: 10.1016/j.cell.2014.05.010. © 2014 Elseiver Inc. Broader uses in SciTech Since the discovery of the CRISPR/Cas9 system, several applications have been envisioned and applied Generation of transgenic animals by gene editing is routine Similarly, in plants gene editing is a standard lab practice CRISPR components are now readily available on single plasmids (sgRNA, Cas9 and promoter) Thus genome editing is at the easiest it has ever been for eukaryote organisms Cell. 2014 Jun 5;157(6):1262-78. doi: 10.1016/j.cell.2014.05.010. © 2014 Elseiver Inc.  Inhibition of Cas9 activity Cas9 activity as a nuclease is pivotal to gene editing Yet, its unchecked activity can pose serious consequences to the organism Methods to alter, reduce or eliminate Cas9 activity after a specified period are important to reduce off target effects Inactivation by proteins administered as viral DNA is therefore a potentially Cell. 2017 Sep 7;170(6):1224-1233.e15. doi: 10.1016/j.cell.2017.07.037. interesting avenue of attenuation © 2017 Elseiver Inc. Mechanicm of Cas9 inhibition These proteins can target one of the DNA binding and cleavage sites of Cas9 Alteration/occlusion of the active site of Cas9 (HNH) has a dramatic effect on catalytic activity AcrIIC1 is able to effectively interact and occlude the active site of Cas9 Stable interactions maintain a tight link between the proteins thus Cell. 2017 Sep 7;170(6):1224-1233.e15. doi: 10.1016/j.cell.2017.07.037. © 2017 Elseiver Inc. preventing activity of Cas9 Jennifer Doudna © 2017 UC Regents  Double nickase activity Cell. 2013 Sep 12;154(6):1380-9. doi: 10.1016/j.cell.2013.08.021. Instead of DSB cleavage at a single site, nicking at two sites for a staggered break Specific mutation of Cas9 catalytic sites has allowed 2 sgRNA to be used to convey great specificity As a result, single nicking of each strand at a similarly predefined locus reduces the chance of off target effects significantly CRISPR base editors offer more control Engineered by combining several proteins Base editors allow scientists to change a single base only Enabling AA changes without inducing indels Trends in Biotechnology 2019 37, 1121-1142DOI: (10.1016/j.tibtech.2019.03.008) Copyright © 2019 Elsevier Ltd Terms and Conditions Not inducing indels and unspecific indels makes the technique more precise Cytosine base editor (CBE) protein detail Both the gRNA and Cas9/Cpf1 target the machinery to the specific DNA sequence A cytosine deaminase is linked to the corresponding Cas9 or Cpf1 and converts the CàU The protein UGI also attached to the Cas9/Cpf1 effectively overrides Trends in Biotechnology 2019 37, 1121-1142DOI: (10.1016/j.tibtech.2019.03.008) the cells BER system Copyright © 2019 Elsevier Ltd Terms and Conditions Base editors are less error prone HDR is not very efficient (0.5% ) and results in 4.3% indels While a single base editor, such as CBE has a 37% efficiency and only induces 1.1% of indels These base editors can use either Cas9 (dCas9 or nCas9) or Cpf1 Trends in Biotechnology 2019 37, 1121-1142DOI: (10.1016/j.tibtech.2019.03.008) Copyright © 2019 Elsevier Ltd Terms and Conditions What can be gained by using base editors Trends in Biotechnology 2019 37, 1121-1142DOI: (10.1016/j.tibtech.2019.03.008) Copyright © 2019 Elsevier Ltd Terms and Conditions Learning objectives Define the importance of the CRISPR/Cas system in bacterial 1. immunity 2. systems Identify the components of the CRISPR/Cas9 and Cpf1 Describe in detail the mechanism of action of CRISPR/Cas9 in 3. genome editing Discuss potential applications of CRISPR/Cas9 to biology and 4. biotechnology  References (Figures) Figures and Reading Molecular Cell Biology, 8th Ed. (2016) Lodish, H., Berk, A., et al. W. H. Freeman and Company References Articles (Figures and reading) The Heroes of CRISPR. Cell. 2016 Jan 14;164(1-2):18-28. doi: 10.1016/j.cell.2015.12.041. CRISPR-based technologies: prokaryotic defense weapons repurposed. Trends Genet. 2014 Mar;30(3):111-8. doi: 10.1016/j.tig.2014.01.003. Biology and Applications of CRISPR Systems: Harnessing Nature's Toolbox for Genome Engineering. Cell. 2016 Jan 14;164(1-2):29-44. doi: 10.1016/j.cell.2015.12.035. Development and applications of CRISPR-Cas9 for genome engineering. Cell. 2014 Jun 5;157(6):1262- 78. doi: 10.1016/j.cell.2014.05.010. A Broad-Spectrum Inhibitor of CRISPR-Cas9. Cell. 2017 Sep 7;170(6):1224-1233.e15. doi: 10.1016/j.cell.2017.07.037. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell. 2013 Sep 12;154(6):1380-9. doi: 10.1016/j.cell.2013.08.021.