实验生物学_细胞基因操作

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1987: DNA repeat arrays in the intergenic region adjacent to the alkaline phosphatase (iap) gene in Escherichia coli K12. 2002: coining of the CRISPR acronym. 2005: the hypervariable spacers showed sequence homology to viruses or plasmids and hypothesized that CRISPRs and associated proteins could play a role in immunity against transmissible genetic elements.
Cascade pre-crRNA
c
Type II
Cas9 RNase III pre-crRNA
d
Type III
Cas6 pre-crRNA
tracrRNA
Cmr/Cas10 Csm/Cas10 crRNA biogenesis
Intermediate crRNA Intermediate crRNA Mature crRNA
TALEN Vs. ZFN
DeFrancesco L. Nature Biotechnology 2011, 29(8): 681-684
Transcription activator- like effector nuclease (TALEN)
Pros ◆ Highly programmable Cons ◆ Hard to make and keep plasmids ◆ High efficiency ◆ Not suitable for high throughput ◆ Minimal off-targeting cleavage screening
Cas3
Mature crRNA Mature crRNA
PAM RuvC
Bhaya D, et al. Annu. Rev. Genet. 2011. 45:273–97
H11-Marraffini
CRISPR Provides Acquired Resistance Against Viruses in Prokaryotes
ARI 14 July 2015 9:37
Chapter 1: Purpose and principle of cellular genome editing
The purpose of cellular genome editing
◆ Gene knockout ◆ Gene knockin ◆ Gene correction
Why knock out a gene?
aBaidu Nhomakorabea
Cas1/ Cas2 CRISPR Leader/promoter Repeat Spacer Leader/promoter cas cas cas genes cas cas Adaptation
b
Type I
Cascade pre-crRNA
c
Type II
Cas9 RNase III pre-crRNA
With off-target cleavage Minimal off-target cleavage No immunogenicity May have immunogenicity
DeFrancesco L. Nature Biotechnology 2011, 29(8): 681-684
Transcription activator- like effector nuclease (TALEN)
Miller et al. Nature Biotechnology, 2011, 29: 143-148.
TALEN Vs. ZFN
ZFNs Recognize a nucleotide triplet Target specific sequence Context dependence TALENs Recognize single nucleotides Target any sequence Context independence
Zinc-finger nuclease (ZFN)
Adapted from Urnov et al. 2010
◆ Artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain
Keith Joung, OPEN (Oligomerized Pool Engineering)
Zinc-finger nuclease (ZFN)
Pros ◆ Programmable Cons ◆ Hard to find ZFA
◆ High efficiency
◆ Off-targeting cleavage
Clustered regulatory interspaced short palindromic repeat (CRISPR)/ CRISPR associated protein (Cas)-based RNA-guided DNA endonucleases (CRISPR-Cas)
Discovery of CRISPR/Cas system
geted DNA double-stran genome editing through Subsequently, Carroll a potential of designer nu for efficient, locus-spec Moreover, it was shown ogy repair template that deletion mutations (inde end-joining (NHEJ) repa 2002). These early geno induced HR and NHEJ and precise modification To achieve effective g specific DNA DSBs, fou binding proteins have b derived from microbial 2006), zinc finger (ZF) nu tion factors (Urnov et al. activator-like effectors (Christian et al., 2010; M cou and Bogdanove, 20 DNA endonuclease Cas9 mune system CRISPR (C Meganuclease, ZF, an DNA sequences throug meganucleases integra
Knockout with DNA endonuclease
(Double Strand Break)
(Non-Homologous End Joining)
Indel (Insertion or deletion)
Knockout with DNA endonuclease
Pros ◆ High efficiency (50%) ◆ No cell type limitation ◆ Labor saving ◆ Money saving ◆ Encouraging ☺ Cons ◆ Off-targeting
Why knock in a gene?
Conventional knockin method
Conventional knockin method
Cons ◆ Low efficiency (10-6) ◆ Cell type limitation ◆ Labor consuming ◆ Money consuming Pros ◆ High precision
Cell. 2014 Jun 5;157(6):1262-78. doi: 10.1016/j.cell.2014.05.010
Figure 1. Applications of Genome Engineering
Genetic and epigenetic control of cells with genome engineering technologies is enabling a broad range of applications from basic biology to biotechnology and medicine. (Clockwise from top) Causal genetic mutations or epigenetic variants associated with altered biological function or disease phenotypes can
Cellular genome editing
Jia Chen School of Life Science and Technology, ShanghaiTech University
Genome editing is getting hotter and hotter
What can we do with genome editing?
Conventional knockout method
Conventional knockout method
Cons ◆ Low efficiency (10-6) ◆ Cell type limitation ◆ Labor consuming ◆ Money consuming ◆ Disappointing ☹ Pros ◆ High precision
Knockin with DNA endonuclease
Knockin with DNA endonuclease
Pros ◆ Relatively high efficiency (10-4) Cons ◆ Off-targeting
Why correct a gene mutation?
Meganuclease
María José Marcaida et al. PNAS 2008;105:16888-16893
Meganuclease
Pros ◆ Very high specificity Cons ◆ Recognize limited target sites ◆ Hard to modify the enzyme ◆ Have never been widely used
d
Type III
Cas6 pre-crRNA
Annu Rev Microbiol. 2015 Jul 22. [Epub ahead of print]
Repeat Spacer
Mechanism of CRISPR/Cas system
Leader/promoter cas cas
b
Type I
Zinc Finger Array (ZFA)
Adapted from Urnov et al. 2010
◆ However, finding efficient Zinc Finger Array (ZFA) is hard!
Patented Vs. Open recourse
Sangamo BioSciences
Gene correction with CRISPR/Cas9
Chapter 2: Genome editing tools
Generation of genome editing tools
◆ Meganuclease ◆ Zinc-finger nuclease (ZFN) ◆ Transcription activator- like effector nuclease (TALEN) ◆ RNA- guided endonuclease (CRISPR-Cas)