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RISS 인기검색어
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- 저자 : Gopalappa, R. (검색결과 4 건)
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Paired D10A Cas9 nickases are sometimes more efficient than individual nucleases for gene disruption
Gopalappa, Ramu,Suresh, Bharathi,Ramakrishna, Suresh,Kim, Hyongbum ,(Henry) Oxford University Press 2018 Nucleic acids research Vol.46 No.12
<P><B>Abstract</B></P><P>The use of paired Cas9 nickases instead of Cas9 nuclease drastically reduces off-target effects. Because both nickases must function for a nickase pair to make a double-strand break, the efficiency of paired nickases can intuitively be expected to be lower than that of either corresponding nuclease alone. Here, we carefully compared the gene-disrupting efficiency of Cas9 paired nickases with that of nucleases. Interestingly, the T7E1 assay and deep sequencing showed that on-target efficiency of paired D10A Cas9 nickases was frequently comparable, but sometimes higher than that of either corresponding nucleases in mammalian cells. As the underlying mechanism, we found that the HNH domain, which is preserved in the D10A Cas9 nickase, has higher activity than the RuvC domain in mammalian cells. In this study, we showed: (i) the <I>in vivo</I> cleavage efficiency of the HNH domain of Cas9 in mammalian cells is higher than that of the RuvC domain, (ii) paired Cas9 nickases are sometimes more efficient than individual nucleases for gene disruption. We envision that our findings which were overlooked in previous reports will serve as a new potential guideline for tool selection for CRISPR-Cas9-mediated gene disruption, facilitating efficient and precise genome editing.</P>
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Ramu Gopalappa,송명재,아룬판디안찬드라세카란,Soumyadip Das,sabahaq,고현철,Suresh Ramakrishna 대한약학회 2018 Archives of Pharmacal Research Vol.41 No.9
Targeted genome editing by clustered regularly interspaced short palindromic repeats (CRISPR-Cas9) raised concerns over off-target effects. The use of doublenicking strategy using paired Cas9 nickase has been developed to minimize off-target effects. However, it was reported that the efficiency of paired nickases were comparable or lower than that of either corresponding nuclease alone. Recently, we conducted a systematic comparison of the efficiencies of several paired Cas9 with their corresponding Cas9 nucleases and showed that paired D10A Cas9 nickases are sometimes more efficient than individual nucleases for gene disruption. However, sometimes the designed paired Cas9 nickases exhibited significantly lower mutation frequencies than nucleases, hampering the generation of cells containing paired Cas9 nickase-induced mutations. Here we implemented IRES peptide-conjugation of fluorescent protein to Cas9 nickase and subjected for fluorescence-activated cell sorting. The sorted cell populations are highly enriched with cells containing paired Cas9 nickase-induced mutations, by a factor of up to 40-fold as compared with the unsorted population. Furthermore, gene-disrupted single cell clones using paired nickases followed by FACS sorting strategy were generated highly efficiently, without compromising with its low off-target effects. We envision that our fluorescent protein coupled paired nickase-mediated gene disruption, facilitating efficient and highly specific genome editing in medical research.
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Lee, Hyeon J,Gopalappa, Ramu,Sunwoo, Hongjae,Choi, Seo-Won,Ramakrishna, Suresh,Lee, Jeannie T,Kim, Hyongbum H,Nam, Jin-Wu Oxford University Press 2019 Nucleic acids research Vol.47 No.8
<P><B>Abstract</B></P><P>The <I>XIST</I> RNA is a non-coding RNA that induces X chromosome inactivation (XCI). Unlike the mouse <I>Xist</I> RNA, how the human <I>XIST</I> RNA controls XCI in female cells is less well characterized, and its functional motifs remain unclear. To systematically decipher the XCI-involving elements of <I>XIST</I> RNA, 11 smaller <I>XIST</I> segments, including repeats A, D and E; human-specific repeat elements; the promoter; and non-repetitive exons, as well as the entire <I>XIST</I> gene, were homozygously deleted in K562 cells using the Cas9 nuclease and paired guide RNAs at high efficiencies, followed by high-throughput RNA sequencing and RNA fluorescence <I>in situ</I> hybridization experiments. Clones containing <I>en bloc</I> and promoter deletions that consistently displayed no <I>XIST</I> RNAs and a global up-regulation of X-linked genes confirmed that the deletion of <I>XIST</I> reactivates the inactive X chromosome. Systematic analyses of segmental deletions delineated that exon 5 harboring the non-repeat element is important for X-inactivation maintenance, whereas exons 2, 3 and 4 as well as the other repeats in exon 1 are less important, a different situation from that of mouse <I>Xist</I>. This Cas9-assisted dissection of <I>XIST</I> allowed us to understand the unique functional domains within the human <I>XIST</I> RNA.</P>
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Somatic Mutations in TSC1 and TSC2 Cause Focal Cortical Dysplasia
Lim, J.S.,Gopalappa, R.,Kim, S.H.,Ramakrishna, S.,Lee, M.,Kim, W.i.,Kim, J.,Park, S.M.,Lee, J.,Oh, J.H.,Kim, H.D.,Park, C.H.,Lee, J.S.,Kim, S.,Kim, D.S.,Han, J.M.,Kang, H.C.,Kim, H.(.,Lee, J.H. University of Chicago Press [etc.] 2017 American journal of human genetics Vol.100 No.3
<P>Focal cortical dysplasia (FCD) is a major cause of the sporadic form of intractable focal epilepsies that require surgical treatment. It has recently been reported that brain somatic mutations in MTOR account for 15%-25% of FCD type II (FCDII), characterized by cortical dyslamination and dysmorphic neurons. However, the genetic etiologies of FCDII-affected individuals who lack the MTOR mutation remain unclear. Here, we performed deep hybrid capture and amplicon sequencing (read depth of 100 x-20,012 x) of five important mTOR pathway genes-PIK3CA, PIK3R2, AKT3, TSC1, and TSC2-by using paired brain and saliva samples from 40 FCDII individuals negative for MTOR mutations. We found that 5 of 40 individuals (12.5%) had brain somatic mutations in TSC1 (c.64C>T [p.Arg22Trp] and c.610C>T [p.Arg204Cys]) and TSC2 (c.4639G>A [p.Va11547I1e]), and these results were reproducible on two different sequencing platforms. All identified mutations induced hyperactivation of the mTOR pathway by disrupting the formation or function of the TSC1TSC2 complex. Furthermore, in utero CRISPR-Cas9-mediated genome editing of Tsc1 or Tsc2 induced the development of spontaneous behavioral seizures, as well as cytomegalic neurons and cortical dyslamination. These results show that brain somatic mutations in TSC1 and TSC2 cause FCD and that in utero application of the CRISPR-Cas9 system is useful for generating neurodevelopmental disease models of somatic mutations in the brain.</P>
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