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CRISPR as a strong gene editing tool
( Shengfu Shen ),( Tiing Jen Loh ),( Hongling Shen ),( Xuexiu Zheng ),( Haihong Shen ) 생화학분자생물학회(구 한국생화학분자생물학회) 2017 BMB Reports Vol.50 No.1
Clustered regularly-interspaced short palindromic repeats (CRISPR) is a new and effective genetic editing tool. CRISPR was initially found in bacteria to protect it from virus invasions. In the first step, specific DNA strands of virus are identified by guide RNA that is composed of crRNA and tracrRNA. Then RNAse III is required for producing crRNA from pre-crRNA. In The second step, a crRNA:tracrRNA:Cas9 complex guides RNase III to cleave target DNA. After cleavage of DNA by CRISPR-Cas9, DNA can be fixed by Non- Homologous End Joining (NHEJ) and Homology Directed Repair (HDR). Whereas NHEJ is simple and random, HDR is much more complex and accurate. Gene editing by CRISPR is able to be applied to various biological field such as agriculture and treating genetic diseases in human. [BMB Reports 2017; 50(1): 20-24]
SR proteins regulate V6 exon splicing of CD44 pre-mRNA
( Tiing Jen Loh ),( Heegyum Moon ),( Ha Na Jang ),( Yongchao Liu ),( Namjeong Choi ),( Shengfu Shen ),( Darren Reece Williams ),( Da-woon Jung ),( Xuexiu Zheng ),( Haihong Shen ) 생화학분자생물학회(구 한국생화학분자생물학회) 2016 BMB Reports Vol.49 No.11
CD44 pre-mRNA includes 20 exons, of which exons 1-5 (C<sub>1</sub>-C<sub>5</sub>) and exons 16-20 (C<sub>6</sub>-C<sub>10</sub>) are constant exons, whereas exons 6-15 (V<sub>1</sub>-V<sub>10</sub>) are variant exons. V6-exon-containing isoforms have been known to be implicated in tumor cell invasion and metastasis. In the present study, we performed a SR protein screen for CD44 V<sub>6</sub> splicing using overexpression and lentivirus-mediated shRNA treatment. Using a CD44 V<sub>6</sub> minigene, we demonstrate that increased SRSF3 and SRSF4 expression do not affect V<sub>6</sub> splicing, but increased expression of SRSF1, SRSF6 and SRSF9 significantly inhibit V<sub>6</sub> splicing. In addition, using a constitutive exon-specific primer set, we could not detect alterations of CD44 splicing after SR protein-targeting shRNA treatment. However, using a V<sub>6</sub> specific primer, we identified that reduced SRSF2 expression significantly reduced the V6 isoform, but increased V<sub>6-10</sub> and V<sub>6,8-10</sub> isoforms. Our results indicate that SR proteins are important regulatory proteins for CD44 V<sub>6</sub> splicing. [BMB Reports 2016; 49(11): 612-616]