Natronobacterium gregoryi Argonaute Feng Gao Xiao Z Shen Feng Jiang Yongqiang Wu amp Chunyu Han Gene E diting Tools Overview httpsenwikipediaorgwikiGenomeediting In order to do a targeted genome editing we need to make a double stranded breaks at specific sites w ID: 1047073
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1. DNA-guided genome editing using the Natronobacterium gregoryi ArgonauteFeng Gao, Xiao Z Shen, Feng Jiang, Yongqiang Wu & Chunyu Han
2. Gene Editing Tools Overviewhttps://en.wikipedia.org/wiki/Genome_editingIn order to do a targeted genome editing, we need to make a double stranded breaks at specific sites within genomeExisting technologies:MeganucleasesTALENZFNCRISPR-Cas9http://www.the-scientist.com/?articles.view/articleNo/45119/title/-Heroes-of-CRISPR--Disputed/http://www.nature.com/nature/journal/v534/n7605/full/534037a.html
3. CRISPRhttps://biotechconnectla.files.wordpress.com/2015/06/crispr-cas9-figure-1.jpg
4. Relevancy in Gene Editinghttps://www.neb.com/tools-and-resources/feature-articles/crispr-cas9-and-targeted-genome-editing-a-new-era-in-molecular-biology
5. CRISPR vs NgAgo-gDNACRISPRTemplate of ~20bpRequires gRNA, CAS 9 and PAM30-60% efficacy in KDs1-15% efficacy in KIsVariable % off-target mutantsNgAgo-gDNATemplate of ~24bpTemplate must be phosphorylated at the 5’ endLow % of off target mutants
6. Natronobacterium gregoryi ArgonauteNatronobacterium gregori : An halophilic and alkiphilic bacteria that grows at 37°C.Argonaute : a family of endonucleases that require a 5’pSSDNA guideNatronobacterium gregoryi, DSM 3393 (EM from M. Rohde, HZI)
7. Why N. gregoryi?Other species with endonuclease Argonaute protein: Thermus thermophilusPyrococcus furiosus
8. First things first: Does it work in vivo?+Rnase A+Dnase I+Proteinase K
9. First things first: Does it work in vivo?Cleavage at 37°CCan only cleave with guide (either FW or RV), could not cleave with non-complementary guide
10. First things first: Does it work in vivo?Specificity Experiment-NgAgo can only use a 5’ phosphorylated single strand DNA guide.
11. What about off-target cleavage?
12. Are you sure it’s an endonuclease?
13. Is it better than CRISPR?When using CRISPR, the guides are labeled sgRNA for “Short Guide RNA”Normally expresses green fluorescent protein
14. OptimizationSequence LengthSequence
15. 400bp 100bp% of mutantT7E3 Assay
16. DYRK1A (Dual-Specificity Tyrosine-(Y)-Phosphorylation Regulated Kinase 1A) gene. Diseases associated with DYRK1A include microcephaly and seizure disorder. Extensively used in CRISPR.G5: 31%G6: 34%G10: 41%G12: 27%G13: 39%Figure 4a. Silencing human DYRK1A gene
17. EMX1: 24.5%GRIN2B: 26.2%GATA4: 24.8%HBA2: 29%Figure 4b. Silencing other human genes
18. 293T: 20.3%MCF7: 13.7%K562: 24.8%HeLa: 11.2%Figure 4c. Silencing DYRK1A gene in other human cell lines
19. Single mismatch of NT 8 completely abolish its functionSingle mismatch of NT 9-11 severely affects its functionThree consecutive mismatches anywhere completely abolish its function Figure 4d. Determining critical NT in guide G10
20. NgAgo: 29.7-34.4%CRISPR: 31.2-33%Figure 4e. NgAgo is comparable to CRISPR in silencing DYRK1A gene
21. HBA2:NgAgo: 37.6%CRISPR: 0%GATA4:NgAgo: 31.5%CRISPR: 13.1%Figure 4f. NgAgo is better than CRISPR in silencing HBA2 and GATA4 (GC rich) genes
22. Facilitate selection of mutant??
23. CRISPR vs NgAgo-gDNACRISPRTemplate of ~20bpRequires gRNA, CAS 9 and PAM30-60% efficacy in KDs1-15% efficacy in KIsVariable % off-target mutantsNgAgo-gDNATemplate of ~24bpTemplate must be phosphorylated at the 5’ endLow % of off target mutants
24. Supplementary Figure 9Full-length gel images (Unrelated lanes are marked with cross).a, for Fig 1a：Nucleic acids associated with NgAgo in E.coli.b, for Fig 1b: The in vitro plasmid cleavage assay(E.coli.-derived NgAgo).c, for Fig 1c: The in vitro plasmid cleavage assay(E.coli.-derived NgAgo, guides with or without 5' phosphorylation). d, for Fig 2a.e, for Fig 2b.f, for Fig 2c.g, for Fig 3a: The in vitro plasmid cleavage assay (293T cell-derived NgAgo).h, for Fig 3c: western blot (GFP,ACTIN).i, for Fig 3d: western blot (GFP,ACTIN).j, for Fig 4a: T7E1 (DYRK1A) .k, for Fig 4b: T7E1 (DYRK1A,EMX1,GRIN2B,GATA4,HBA2).Research ethics
25. Summary of Dal-iGEM project4 microbiome samples submitted for sequencing2-4 bacterial strains isolated from cellulose plates2nd trip to park for fecal samples from more animals and plantsa. Microbiome profilingb. Metagenomic sequencingPlating for more isolates that can utilize celluloseSecondary screen of isolates that can detoxify sapc. Genome sequencing of an ideal bacterial strain that could utilized cellulose and detoxify sapCloning of the genes that are responsible for the phenotypesCloning of NgAgo into a broad-range bacterial vector and deposit in BioBrick