1 Feng Gao, Xiao Z Shen, Feng Jiang, Yongqiang Wu & Chunyu HanDNA-guided genome editing using the Natronobacterium gregoryi Argonaute Feng Gao, Xiao Z Shen, Feng Jiang, Yongqiang Wu & Chunyu Han
2 Gene Editing Tools Overviewhttps://en.wikipedia.org/wiki/Genome_editing In order to do a targeted genome editing, we need to make a double stranded breaks at specific sites within genome Existing technologies: Meganucleases TALEN ZFN CRISPR-Cas9 of-CRISPR--Disputed/ Gene editing in eukaryotics has proven difficult, and although many tools have been developed, they often have low efficiency and low accuracy. Never the less these are important tools in genetics and many other areas of research in order to see how different genes act and react.
3 CRISPR Clustered regularly interspaced palindromic repeats (CRISPR) CRISPR is a bacterias adaptive immune system. The bacteria has what dr. Rohde calls a “trophy shelve” of fragmented DNA that is has taken from intruders and incorporated into its genome as CRISPR. When this area of genome is transcribed it is then cleaved at the (Trans-activating) tracrRNA sites as seen here. This “crisprRNA” is then incorporated into the enzyme Cas9 and used as a template for any intruder with the same sequence. The reason CAS9 doesn’t cleave it’s own genome is because foreign DNA contains a protospacer-adjacent motif or ‘PAM’ sequence that the cr RNA recognizes as non-self. It must follow immediately on the 3’ side of the crRNA https://biotechconnectla.files.wordpress.com/2015/06/crispr-cas9-figure-1.jpg
4 Relevancy in Gene EditingWith the discovery of the Type II CRIPSR/CAS 9 system researchers began creating hybrids of cRNA and trRNA which is called guideRNA so that they can target specific gene sequences (~20bp). CAS9 will cleave both strands of DNA resulting in the DS DNA repair mechanisms to kick in and cause an insertion/deletion. This can cause knockdowns or if donor dna (ei dna that you want to be incorporated) is around, homology directed repair can allow for knock-ins. You must be sure there is a NGG sequence right before the gene of interest (on 3’ https://www.neb.com/tools-and-resources/feature-articles/crispr-cas9-and-targeted-genome-editing-a-new-era-in-molecular-biology https://www.neb.com/tools-and-resources/feature-articles/crispr-cas9-and-targeted-genome-editing-a-new-era-in-molecular-biology
5 CRISPR vs NgAgo-gDNA CRISPR NgAgo-gDNA Template of ~20bpRequires gRNA, CAS 9 and PAM 30-60% efficacy in KDs 1-15% efficacy in KIs Variable % off-target mutants NgAgo-gDNA Template of ~24bp Template must be phosphorylated at the 5’ end Low % of off target mutants CRISPR -the protospacer sequence can deal with 5 mismatches, PAM can deal with 1 -therefore can often create off-target mutants which are only detectable through full genome screens. Figure B in previous slide is a way to deal with off target mutants. -can be less useful on sequences high or low in GC content -Produces relatively okay results for KDs (30%-60%) -gRNA tend to form secondary structures on itself Ng-Ago -Very little off-target activity because there aren’t many 5’ ppssdna found intracellular
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 guide Endonucleases: cleaves some where in the middle of nucleic acid Argonautes are big players in RNA silencing processes such as RNAi, miRNA, siRNA Argonautes are found in nearly all organisms, not just prokaryotes. Like in the RISC complex in humans! Photo: Natronobacterium gregoryi, DSM 3393 (EM from M. Rohde, HZI)
7 Why N. gregoryi? Other species with endonuclease Argonaute protein:Thermus thermophilus Pyrococcus furiosus Many other suitable argonaute protein have been found in bacteria, but the problem was that they were useless as physiological temperatures. The ones mentioned above only cleave at above 65C. In order to find a suitable candidate, they used the amino acid sequences of T.thermophilus and P. furiosus argonaute proteins and used PSI-BLAST (Position-Specific Iterative Basic Local Alignment Search Tool) against the NCBI non-redundant protein data base. This program is used to find distant relatives of a protein. First, a list of all closely related proteins is created. These proteins are combined into a general "profile" sequence, which summarises significant features present in these sequences. A query against the protein database is then run using this profile, and a larger group of proteins is found. This larger group is used to construct another profile, and the process is repeated. By including related proteins in the search, PSI-BLAST is much more sensitive in picking up distant evolutionary relationships than a standard protein-protein BLAST. They used the amino acid sequence of TtAgo and PfAgo and found that NgAgo was a possible candidate.
8 First things first: Does it work in vivo?They expressed NgAgo in Ecoli, which produce both ssRNA and ssDNA in order to see which guides the protein uses. They isolated NgAgo, processed with proteinase K and then with either RNaseA or Dnase I to see which guide the protein picked up. +Proteinase K +Rnase A +Dnase I
9 First things first: Does it work in vivo?Cleavage at 37°C Can only cleave with guide (either FW or RV), could not cleave with non-complementary guide They designed target guides, both forward and reverse, to a target site on the plasmid above. They also designed a guide that had a non-complementary sequence to the plasmid. When treated with FW or RV, they were able to create an open circle plasmid, when both were added a linear species was seen. They had to heat the protein up to 55°𝐶 in order to switch out the endogenous guide for the designed one.
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?Three different experiments were done in order to confirm specificity to the target sequence in the guide. They transfected HEK (human embryonic kidney) 293T cells. This cell line contains Large T antigen from SV40 (simian virus). This cell line gives high transfection rates. They co-transfected there plasmid with NgAgo and different types of guides. As seen before only 5’-p-ssDNA was isolated from purified NgAgo. Interestingly, when only the plasmid was transfected, no guide could be isolated from the purified protein suggesting that there is little endogenous 5’p-ssDNA in the cell. They next tested if time played a role in successful guide loading. They postponed the transfection of the 5’p-ssDNAs 12h and 24h after the transfection of the plasmid. Expression of the guide decreased at the 24h time point which means that loading is most successful right after NgAgo is expressed. This experiment was done out of the cell line. NgAgo was isolated from HEK cells and incubated with the guide at 55C and 37C. Even after 8 hours of incubate, unloaded NgAgos could load guides at physiological temperature. This is could because even if there were ssDNAs in the cell, NgAgo would not load them.
12 Are you sure it’s an endonuclease?Target plasmid was incubated with purified NgAgo that was preloaded with a forward guide. This was incubated together for 4h 8h and 72 hours with no further degradation between 8h and 72hr time point suggesting that NgAgo is not an exonuclease. They then sequenced the 72hr product and found that 1-20 nt were removed randomly from the target/guide area. This was doen using sanger sequencing
13 Is it better than CRISPR?Normally expresses green fluorescent protein Is it better than CRISPR? When using CRISPR, the guides are labeled sgRNA for “Short Guide RNA” They created a couple guides for NgAgo against the PEGFP-N1 Plasmid. This plasmid expresses Green Fluorescent protein when inserted into cells. After using either the NgAgo system or the CRISPR/CAS9 system, they lysed the cells for protein and ran that on a western blot and probed with eGFP As you can see from the blots, the NgAgo system had a better KD effiency than the CRISPR/Cas9 system.
14 Optimization Sequence Length SequenceThis is the result of three independent experiments. They created guides of different nucleotide length against GFP and co-transfected them into 293T cells. They then made a cell lysate and ran it on a western blot and probed fro GFP. It is quite clear from the blots the a 24nt guide works most efficiently
15 400bp bp T7E3 Assay % of mutant
16 Figure 4a. Silencing human DYRK1A geneDYRK1A (Dual-Specificity Tyrosine-(Y)-Phosphorylation Regulated Kinase 1A) gene. Diseases associated with DYRK1A include microcephaly and seizure disorder. Extensively used in CRISPR.
17 Figure 4b. Silencing other human genesEMX1: 24.5% GRIN2B: 26.2% GATA4: 24.8% HBA2: 29%
18 Figure 4c. Silencing DYRK1A gene in other human cell linesMCF7: 13.7% K562: 24.8% HeLa: 11.2%
19 Figure 4d. Determining critical NT in guide G10Single mismatch of NT 8 completely abolish its function Single mismatch of NT 9-11 severely affects its function Three consecutive mismatches anywhere completely abolish its function
20 Figure 4e. NgAgo is comparable to CRISPR in silencing DYRK1A gene
21 Figure 4f. NgAgo is better than CRISPR in silencing HBA2 and GATA4 (GC rich) genes
22 Facilitate selection of mutant??
23 CRISPR vs NgAgo-gDNA CRISPR NgAgo-gDNA Template of ~20bpRequires gRNA, CAS 9 and PAM 30-60% efficacy in KDs 1-15% efficacy in KIs Variable % off-target mutants NgAgo-gDNA Template of ~24bp Template must be phosphorylated at the 5’ end Low % of off target mutants CRISPR -the protospacer sequence can deal with 5 mismatches, PAM can deal with 1 -therefore can often create off-target mutants which are only detectable through full genome screens. Figure B in previous slide is a way to deal with off target mutants. -can be less useful on sequences high or low in GC content -Produces relatively okay results for KDs (30%-60%) -gRNA tend to form secondary structures on itself Ng-Ago -Very little off-target activity because there aren’t many 5’ ppssdna found intracellular
24 Research ethics 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).
25 Summary of Dal-iGEM project4 microbiome samples submitted for sequencing 2-4 bacterial strains isolated from cellulose plates 2nd trip to park for fecal samples from more animals and plants a. Microbiome profiling b. Metagenomic sequencing Plating for more isolates that can utilize cellulose Secondary screen of isolates that can detoxify sap c. Genome sequencing of an ideal bacterial strain that could utilized cellulose and detoxify sap Cloning of the genes that are responsible for the phenotypes Cloning of NgAgo into a broad-range bacterial vector and deposit in BioBrick