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1 Department of Chemistry, Seoul National University, Seoul 08826, Republic of Korea.
2 Center for Genome Engineering, Institute for Basic Science, Seoul 08826, Republic of Korea.
Daesik Kim et al.
Genome Res .
2018 Dec .
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1 Department of Chemistry, Seoul National University, Seoul 08826, Republic of Korea.
2 Center for Genome Engineering, Institute for Basic Science, Seoul 08826, Republic of Korea.
Effects of chromatin structure on Cas9 editing efficiency. ( A ) Schematic overview of the method for investigating the effects of chromatin structure on Cas9 editing efficiency. ( B ) Representative IGV images obtained using ENCODE DNase-seq data at two on-target sites with the same Cas9 target sequence present in both open and closed chromatin regions. ( C ) Relative fractions of intact genomic DNA not cleaved by DNase I measured using real-time quantitative PCR at the Cas9 target site in HeLa cells. The range 0–32U denotes the concentration of DNase I. Error bars, SEM from at least three independent experiments. ( D ) SpCas9-induced mutation frequencies at 12 pairs of endogenous target sites with the same DNA sequence present in both open and closed chromatin regions in HEK 293T or HeLa cells. Indel frequencies were measured using targeted deep sequencing. Pairs of Cas9 target sites are represented with dots with different colors. Mean indel frequencies ± SEM are shown ( n = 12 target sites).
Effects of chromatin structure on Cas9 activity at off-target sites. ( A ) Mismatched sgRNAs that differed from the Cas9 target sequence by 1 or 2 nt were targeted to sites in both open (blue) and closed (red) chromatin regions in HEK 293T and HeLa cells. Indel frequencies were measured using targeted deep sequencing. Error bars, SEM ( n = 3). ( B ) Indel frequencies at the on-target sites in open and closed chromatin states using on-/off-target specific sgRNAs in HeLa cells. Indel frequencies were measured using targeted deep sequencing. Error bars, SEM ( n = 3).
DIG-seq using native chromatin DNA. ( A ) Overview of DIG-seq to identify genome-wide Cas9 in vitro cleavage sites using native chromatin DNA. ( B ) A representative IGV image showing a staggered alignment ( top ) and a straight alignment ( bottom ) of whole-genome sequence reads at the on-target site. ( C ) A Venn diagram showing the number of in vitro cleavage sites identified by Digenome-seq using histone-free DNA or by DIG-seq using chromatin DNA with the HBB -targeted CRISPR-Cas9. ( D ) Sequence logos obtained via WebLogo using in vitro cleavage sites captured by Digenome-seq or DIG-seq. ( E ) Scatterplot of DNA cleavage scores at sites captured by Digenome-seq versus DIG-seq. ( F ) Scatterplot of indel frequencies versus DNA cleavage scores at sites captured by DIG-seq using HeLa chromatin DNA. ( G ) Pearson's correlation coefficients ( R -square values) obtained with indel frequencies versus DNA cleavage scores at off-target sites identified by Digenome-seq using chromatin or histone-free DNA. Error bars, SEM ( n = 5). The P -value was calculated by Student's t -test.
Comparison of DIG-seq with other in vitro methods. Venn diagrams showing the number of in vitro cleavage sites identified by DIG-seq, Digenome-seq, CIRCLE-seq, and SITE-seq.
Kim D, Bae S, Park J, Kim E, Kim S, Yu HR, Hwang J, Kim JI, Kim JS.
Kim D, et al.
Nat Methods. 2015 Mar;12(3):237-43, 1 p following 243. doi: 10.1038/nmeth.3284. Epub 2015 Feb 9.
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PMID: 25664545
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Kim D, et al.
Genome Res. 2016 Mar;26(3):406-15. doi: 10.1101/gr.199588.115. Epub 2016 Jan 19.
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To investigate whether and how CRISPR-Cas9 on-target and off-target activities are affected by chromatin in eukaryotic cells, we first identified a series of identical endogenous DNA sequences present in both open and closed chromatin regions and then measured mutation frequencies at these sites in human cells using Cas9 complexed with matched or mismatched sgRNAs. Unlike matched sgRNAs, mismatched sgRNAs were highly sensitive to chromatin states, suggesting that off-target but not on-target DNA cleavage is hindered by chromatin. We next performed Digenome-seq using cell-free chromatin DNA (now termed DIG-seq) and histone-free genomic DNA in parallel and found that only a subset of sites, cleaved in histone-free DNA, were cut in chromatin DNA, suggesting that chromatin can inhibit Cas9 off-target effects in favor of its genome-wide specificity in cells.
© 2018 Kim and Kim; Published by Cold Spring Harbor Laboratory Press.
Effects of chromatin structure on Cas9 editing efficiency. ( A ) Schematic overview…
Effects of chromatin structure on Cas9 activity at off-target sites. ( A )…
DIG-seq using native chromatin DNA.…
DIG-seq using native chromatin DNA. ( A ) Overview of DIG-seq to identify…
Comparison of DIG-seq with other in vitro methods. Venn diagrams showing the number…
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The Joy of Sex: The Ultimate Revised Edition Paperback – December 29, 2009
by
Alex Comfort
(Author)
4.5 out of 5 stars
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A revised and updated edition of the book famous for helping couples discover how sex can be playful, erotic, passionate, exhilarating, and most of all, pleasurable. The Joy of Sex revolutionized how we experience our sexuality. An international bestseller since it was first published in 1972, Dr. Alex Comfort’s classic work dared to celebrate the joy of human physical intimacy with such authority and candor that a whole generation felt empowered to enjoy sex. Now fully updated, revised, and reillustrated, The Joy of Sex once again sets the standard as the world’s most trusted sex manual. Substantial revisions from sex expert and relationship psychologist Susan Quilliam include new information on: • Key scientific discoveries in the fields of psychology, physiology, and sexology • The Internet and couple-friendly pornography • The importance of sex to our growth as people and partners • Maintaining a fulfilling sex life as we get older Above all, The Joy of Sex emphasizes the importance of happy and healthy sexuality in our lives.
“Answers questions no one had in 1972 . . . this time through the eyes of women, too.” — New York Times “There’s something to celebrate about the enduring life of so seminal a piece of educational literature.” —NPR “[Succeeds] in bringing The Joy of Sex up to current standards.” — The New Yorker “An old book now has a lot of new tricks.” — Men’s Health “A bellwether of human sexuality for decades . . . the new version is better.” — Washington Post “Witty, fanciful, and mercifully free of moralizing.” — Time “An intelligent sex manual that is serious without being solemn.” —Desmond Morris, author of People Watching and The Human Sexes: The Natural History of a Man and a Woman
DR. ALEX COMFORT was one of the world’s leading experts in the field of human sexuality and one of the most versatile authors of the twentieth century. He died in March 2000 at the age of eighty. SUSAN QUILLIAM is a relationship psychologist and an advice columnist. She writes, broadcasts, and presents internationally on topics surrounding love and sexuality.
On Gourmet Lovemaking All of us, barring any physical limitations, are able to dance and sing — after a fashion. This, if you think about it, summarizes the justification for learning to make love. Love, in the same way as singing, is something to be taken spontaneously. On the other hand, the difference between Pavlova and the Palais de Danse, or opera and barbershop singing, is much less than the difference between sex as our recent ancestors came to accept it and sex as it can be. At least we recognize this now (so that instead of worrying if sex is sinful, most people now worry whether they are “getting satisfaction” — one can worry about anything, given the determination). And there are now enough books about the basics; we are largely past the point of people worrying about the normality, possibility, and variety of sexual experience. This book is slightly different, in that there are now enough people who have those basics and want more depth of understanding, solid ideas, and inspiration. To draw a parallel, chef-grade cooking doesn’t happen naturally: it starts at the point where people know how to prepare and enjoy food, are curious about it and willing to take trouble preparing it, read recipe hints, and find they are helped by one or two techniques. It’s hard to make mayonnaise by trial and error, for instance. Gourmet sex, as we define it, is the same — the extra one can get from comparing notes, using some imagination, trying way-out or new experiences, when one already is making satisfying love and wants to go on from there. This book will likely attract four sorts of readers. First, there are those who don’t fancy it, find it disturbing, and would rather stay the way they are — these should put it down, accept our apologies, and
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