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A New Genome Editing Tool: CRISPR/Cas Systems D2 楊 斌

2013/06/30 16:59 に Takashi Matsuo が投稿

Three decades ago, an unusual segment of neighbouring DNA that consisted of short, directly repeating nucleotide sequences flanked by short unique segments were discovered in Escherichia coli, but the biological significance of these sequences were unknown[1]. Now, these sequences were referred to as CRISPR (clustered regularly interspaced short palindromic repeat)-associated (Cas) systems which was shown to be versatile tool for genome editing [2]. 

The CRISPR-Cas systems involve RNA which contains similar sequences to viruses or plasmids and allow detection of and protection against mobile genetic elements, so that they were believed to provid specific defences against invaders [3]. Several types of CRISPR-Cas systems were identified in which the type II systems use a single endonuclease, Cas9 [2, 4]. Cas9 acts together with two RNA transcripts, CRISPR RNA (crRNA) and trans-acting CRISPR RNA (tracrRNA) to locate and introduce double-stranded breaks in target DNA. To make the system much simple, the dual RNA were reconfigured as a single guided RNA (sgRNA) in gene editing [2].

In this year, 12 papers were published in gene modifications by using engineered Cas9 in bacterial, human cells and zebrafish. For example, in Streptococcus pneumoniae, nearly 100% of cells contained the desired mutation, and in E. coli, 65% contained the mutation [5]. In various human cell lines, the gene-targeting achieved up to 38% success of introduction of double-stranded breaks and was accompanied by only a low level of Cas9 toxicity [6]. Another usage of Cas9 was that of CRISPR interference (CRISPRi), which was used to repress multiple target genes simultaneously, and its effects are reversible [7].

Compared with using zinc finger nucleases (ZFNs) and transcription activator?like effector nucleases (TALENs), CRISPR-Cas systems induced targeted genetic modifications in zebrafish embryos with similar efficiencies [8, 9]. And this approach offers distinct advantages because it relies on simple base pairing to a guide RNA, in contrast to the ZFN and TALEN proteins, which require a new protein to be engineered for each new cleavage site. But it’s also has a week point that sgRNA expression is a limiting factor in the genome editing reactions [10]. Above all, this RNA-guided DNA recognition platform provides a simple approach for selectively perturbing gene expression on a genome-wide scale. 

[1] Ishino, Y., Shinagawa, H., Makino, K., Amemura, M. & Nakata, A. (1987) Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. J. Bacteriol. 169, 5429?5433
[2] Jinek, M. et al. (2012) A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science 337, 816?821
[3] Barrangou, R. et al. (2007) CRISPR provides acquired resistance against viruses in prokaryotes. Science. 315, 1709?1712
[4] Brouns, S. J. et al. (2008) Small CRISPR RNAs guide antiviral defense in prokaryotes. Science. 321, 960?964 
[5] Jiang, W., Bikard, D., Cox, D., Zhang, F. & Marraffini, L. A. (2013) RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature Biotechnol. 31, 233?239 
[6] Cong, L. et al. (2013) Multiplex Genome Engineering Using CRISPR-Cas Systems. Science. 339, 819?823
[7] Qi, L. S. et al. (2013) Repurposing CRISPR as an R NA-Guided Platform for Sequence-Specific Control of Gene Expression. Cell. 152, 1173?1183.
[8] Hwang, W. Y. et al. (2013) Efficient genome editing in zebrafish using a CRISPR-Cas system. Nature Biotechnol. 31, 227?229 
[9] An Xiao et al. (2013) Chromosomal deletions and inversions mediated by TALENs and CRISPR/Cas in zebrafish. Nucleic Acids Research, 2013, 1?11
[10] Jinek, M. et al. (2013) RNA-programmed genome editing in human cells. eLIFE. http://dx.doi.org/10.7554/eLife.00471