Transcript DNA-guided genome editing using the

DNA-guided genome editing using
the Natronobacterium gregoryi
Argonaute
Feng Gao, Xiao Z Shen, Feng Jiang, Yongqiang Wu & Chunyu Han
Gene Editing Tools Overview
 https://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
 http://www.the-scientist.com/?articles.view/articleNo/45119/title/-Heroesof-CRISPR--Disputed/
 http://www.nature.com/nature/journal/v534/n7605/full/534037a.html
CRISPR
https://biotechconnectla.files.wordpress.com/2015/06/crispr-cas9-figure-1.jpg
Relevancy in Gene Editing
https://www.neb.com/tools-and-resources/feature-articles/crispr-cas9and-targeted-genome-editing-a-new-era-in-molecular-biology
CRISPR vs NgAgo-gDNA
CRISPR
- Template of ~20bp
NgAgo-gDNA
- Template of ~24bp
- Requires gRNA, CAS 9 and - Template must be
PAM
phosphorylated at the 5’
end
- 30-60% efficacy in KDs
- Low % of off target mutants
- 1-15% efficacy in KIs
- Variable % off-target
mutants
Natronobacterium gregoryi Argonaute
 Natronobacterium gregori : An halophilic and alkiphilic
bacteria that grows at 37°C.
 Argonaute : a family of endonucleases that require a
5’pSSDNA guide
Natronobacterium gregoryi, DSM 3393 (EM from M. Rohde, HZI)
Why N. gregoryi?
Other species with endonuclease
Argonaute protein:
1. Thermus thermophilus
2. Pyrococcus furiosus
First things first: Does it work in vivo?
+Proteinase K
+Dnase I
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 noncomplementary guide
First things first: Does it work in vivo?
Specificity Experiment
-NgAgo can only use a 5’
phosphorylated single strand
DNA guide.
What about off-target cleavage?
Are you sure it’s an endonuclease?
Normally expresses
green fluorescent
protein
Is it better than CRISPR?
When using CRISPR, the guides are
labeled sgRNA for “Short Guide RNA”
Optimization
Sequence
Length
Sequence
400bp
T7E3 Assay
% of mutant
100bp
Figure 4a. Silencing human DYRK1A
gene
 G5: 31%
 G6: 34%
 G10: 41%
 G12: 27%
 G13: 39%
DYRK1A (Dual-Specificity Tyrosine-(Y)Phosphorylation Regulated Kinase 1A)
gene.
Diseases associated with DYRK1A include
microcephaly and seizure disorder.
Extensively used in CRISPR.
Figure 4b. Silencing other human genes
 EMX1: 24.5%
 GRIN2B: 26.2%
 GATA4: 24.8%
 HBA2: 29%
Figure 4c. Silencing DYRK1A gene in
other human cell lines
 293T: 20.3%
 MCF7: 13.7%
 K562: 24.8%
 HeLa: 11.2%
Figure 4d. Determining critical NT in guide
G10
 Single 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
Figure 4e. NgAgo is comparable to
CRISPR in silencing DYRK1A gene
 NgAgo: 29.7-34.4%
 CRISPR: 31.2-33%
Figure 4f. NgAgo is better than CRISPR in
silencing HBA2 and GATA4 (GC rich)
genes
 HBA2:
NgAgo: 37.6%
CRISPR: 0%
 GATA4:
NgAgo: 31.5%
CRISPR: 13.1%
Facilitate
selection of
mutant
??
CRISPR vs NgAgo-gDNA
CRISPR
- Template of ~20bp
NgAgo-gDNA
- Template of ~24bp
- Requires gRNA, CAS 9 and - Template must be
PAM
phosphorylated at the 5’
end
- 30-60% efficacy in KDs
- Low % of off target mutants
- 1-15% efficacy in KIs
- Variable % off-target
mutants
Research ethics
Supplementary Figure 9
Full-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).
Summary of Dal-iGEM project
• 4 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