分子生物学读书报告 CRISPR-Cas9

STRUCTURE
MECHANISIM
anti-repeat duplex
the remaining tracrRNA bases form stem loops Target sequence heteroduplex forms
recognizes the Cas9 protein
Cas9-sgRNA binary complex target sequence recognizes the
Cas9-sgRNA-target DNA ternary complex
subsequent cleavage
MECHANISIM
MECHANISIM
Cas9-based systems for altering gene sequence or expression.
APPLICATION
CRISPR/Cas9 swenku.baidu.comstem
GENOME EDITIN
•Genome editing technologies, including chemical- and UVinduced mutagenesis, DNA recombinase-mediated gene replacement, zinc-finger nucleases (ZFNs), and transcriptional activator-like effector nuclease (TALEN) systems, have profoundly contributed to both fundamental and clinical advances in biological research (Urnov et al., 2005; Bedell et al., 2012). •Recently, the CRISPR/Cas system, derived from the adaptive immune system of the bacterium Streptococcus pyogenes has dramatically increased our ability to edit the genomes of diverse species (Sapranauskas et al., 2011).
potentially useful for genome engineering applications
(Cong et al., 2013; Wu et al., 2014).
STRUCTURE
The locus structure of CRISPR
The structure of sg RNA
to globally assess the off-target effects of Cas9 nucleases
or paired nickases in any genome of interest.
PROSPRCTIVE
Methods for efficient delivery and expression of
CONTENT
STRUCTURE MECHANISM APPLICATION PROSPRCTIVE
• To date, three types of CRISPR/Cas systems (I, II, and III)
have been identified. Compared with other systems that
杜琳颖 20131580 刘雪蓉 20131592 刘子钰 20131574
mediated indel mutations and toward HDR-driven
alterations remain a priority for development.
ETHICAL CHALLENGES
due to poor understanding of the mechanism, the possible severe consequences to trigger an entire change of spices, and the potential effects on the ecological balance (Bohannon,2015), some arguments against use of this technology have been raised. a paper reporting gene editing in human embryoswas published in the journal Protein & Cell, which raised concerns about the ethics of employing the CRISPR/Cas9 system (Liang et al., 2015). some countries have already restricted CRISPR/Cas9 technology, with some completely banning its use in humans.
CRISPR/Cas system components will undoubtedly need to be optimized for each particular cell-type or organism to be modified. Strategies for shifting the balance away from NHEJ
Genome editing using the CRISPR/Cas9 system
Generation of genetically modified (GM) mouse model of human diseases Genome editing in specific tissues Simultaneous generation of multiple gene mutations Flexible manipulation in epigenome Novel potentiality in RNA editing
With the capacity for easy and convenient genome, epigenome,and RNA editing, CRISPR/Cas9 provides immense capacity to make remarkable progress in biotechnological, basic biological, and medical research fields.
PROSPRCTIVE
Methods for expanding the targeting range of RNA
guided Cas9 will be important for inducing precise HDR or NHEJ events as well as for implementing multiplex strategies, including paired nickases. The field urgently needs to develop unbiased strategies
REFERENCE
[1] 李君,张毅,陈坤玲.CRISPR/Cas 系统：RNA 靶向的基因组定向编 辑新技术[J].遗传 HERDITAS(Beijing),2013(11),35(11): 1265―1273 [2] 方锐,畅飞,孙照霖.CRISPR/Cas9 介导的基因组定点编辑技术*[J]. 生物化学与生物物理学进展,2013,40(8): 691~702 [3] 张智辉.基因组定点编辑技术的研究进展[J].生命科学,2013(7) [4] Yue Mei , Yan Wang , Huiqian Chen.Recent Progress in CRISPR/Cas9 Technology[J].Journal of Genetics and Genomics,2016(43):63-75 [5] Jeffry D Sander ,J Keith Joung.CRIRISPR-Cas systems for editing, regulating and targeting genomes[J].Nature Biotechnology,2014(5)
necessitate multiple Cas proteins, the type II system only
requires a single Cas protein: Cas9. • The Cas9 protein functions in both RNA-guided DNA recognition relying on sgRNA(single guide RNA) formed by crRNA and tracrRNA fusion and cleavage, making it