JOHNS COUNTY (CBSMiami/CNN) – A North Florida high school is under fire for digitally altering the yearbook photos of more than 80 girls in order to cover up their cleavage.
When Bartram Trail High School freshman Riley O’Keefe saw her yearbook photo, she noticed a black bar was added to cover more of her breasts.
“I couldn’t believe that they printed the yearbook looking like that,” O’Keefe, 15, told CNN. “And then I started to flip through the yearbook and saw more and more girls with their chest edited.”
She texted her mother a photo of it on Wednesday from the school in St. Johns, Florida near Jacksonville.
“I know she’s worn (the outfit) in school hundreds of times because it’s like her go-to outfit,” her mother Stephanie Fabre told CNN, saying she believes the outfit had met the district’s dress code. The dress code states that girls’ tops “must cover the entire shoulder and they must be modest and not revealing or distracting.”
There were 80 photos of female students that were altered in the yearbook this year, the district’s Chief of Community Relations Christina Langston said.
The high school’s website has a disclaimer saying that if student portraits in the yearbook did not match the district’s student code of conduct, they may be “digitally adjusted.”
The superintendent said there was an insufficient review before the school decided to edit some of the students’ images. He called the staff member involved an “outstanding educator” and said there will be changes in how the content is considered in yearbooks to come.
The student dress code prohibits clothing that is “immodest, revealing, or distracting,” according to the district’s code of conduct. However, each school’s principal has the “final authority” over whether a student’s dress is appropriate, the code says.
Reviewed: BBL and Moxify Laser Treatment for Sun Damage | Who What Wear
"In the past, BBLs [broadband light therapies] were really painful," said Emer. "They also were really high risk for Asian skin types, and mixed ethnicities, and the risk of developing hyperpigmentation or what we call 'striping' was really high." This treatment was also ideal for me since I didn't have as severe sun damage as someone much older might have. (Emer referred to doing this treatment before things get worse as pre-juvenation.)
BBL and Moxify is a three-part, combination treatment pioneered by Emer that reduces sun damage, discoloration, redness, and wrinkles. It can also be used in other areas, such as the face and neck, and even elbows, and knees—all with less downtime than other laser treatments. It starts off with BBL Hero, which stands for Broadband Light High Energy Rapid Output, and is the newest, most advanced BBL light therapy on the market today. It uses different filters depending on the type of discoloration you have (red, brown) and is fast, treating the area with fast-pulsing high energy within seconds.
The BBL Hero gets followed by Moxi, which is a non-ablative low-density laser used for skin resurfacing and deep stimulation of collagen but doesn't have the downtime needed with more aggressive lasers, like Halo.
The final step is Emer's Aerify Illuminating Mask, along with a daily skincare regimen from his Emerage Cosmetics line that I would follow for up to two weeks. Depending on how much sun damage there is, it could take several treatments for there to be a significant improvement. He says the first two treatments should be done closer together, like four to eight weeks apart, then possibly every three to four months. I was comped for this service, but it usually costs between $850 and $1200.
On the day of my treatment, I didn't have to do anything to prepare beforehand. A numbing solution was applied to my chest. Then came the first step: BBL Hero, which I could barely feel at first, but then started to feel a little warm. It lasted only a couple of minutes, but you could see the redness starting to come out of my skin. Next, it was time for the Moxi laser. This felt like mild stinging, or pinpricks, but still very minimal pain. I was given a small fan to blow cold air onto my chest as it began to feel hot by the end, similar to a sunburn.
By the second and third day, the redness was significantly reduced, and the skin began feeling together. Then, on the fourth day, I began to experience a dull itch, and the skin started to peel. I also noticed the tiny dots were transforming into pin-sized scabs with an almost sandpaper-like texture. On the days that followed, my chest began to really itch and then peel. It was not cute, but I could already see an improvement in how the skin looked and felt, as it was much smoother. It ended up taking about a week and a half for my chest to finally finish peeling.
After two weeks post-treatment, I'd have to say I'm pretty happy with BBL and Moxify. I had a red patch I know was from a Palm Springs trip in 2009 that is much lighter than it was before. An acne scar on my boob that I had gotten during the past year (pandemic breakouts, can anyone relate?) is now less pronounced. But I knew that this would probably not be a one-and-done situation, and more treatments may be needed to see a significant change in my overall chest texture and wrinkles. In the meantime, I will never go a day without sunscreen and promise to only lay by the pool underneath a gigantic umbrella and wearing a turtleneck. As for sleeping on my side, J.Lo allegedly sleeps on her back surrounded by pillows to prevent wrinkles, so I guess that's next on my list!
Nicole Scherzinger flaunts cleavage in plunging suit jacket with nothing underneath - Daily Star
The singer showed off the outfit on social media and captioned the post with a single cheeky peach emoji.
The silk number, which was ruched up on the sleeves, had fine buttons on the cuffs and one button in the middle of the jacket, which appeared to be vital in preventing the star from flashing her ample assets.
Nicole, 42, wore her long black hair straight and sleek and allowed some to fall down past her shoulders onto her chest, as well as rest behind her back.
She paired the look with sparkling jewellery and several necklaces, including a delicate chain that hugged her cleavage in the shot.
She chose long dangly earrings to compliment the outfit and a simple yet sultry makeup look with a pink and glossy lip for her pout.
As well as a series of snaps, she shared a looping video of her flicking her hair back and cheekily flashes a smile to the camera to her 4.9million followers.
There's MUCH more where that came from! Want all the jaw-dropping stories from the world of showbiz and up to the minute news from TV and soaps?
We'll bring you the inside track from telly expert Ed Gleave and soap specialist Sasha Morris. Oh, and your daily fix of Piers, Katie Price, Demi Rose and all your other Daily Star favs.
The 42-year-old looked stunning in a pink and white print two-piece bikini with the setting sun slowly moving behind her amongst the waves.
Parents, students angered after 80 female students' yearbook photos are altered to mask
Flipping open a yearbook for the first time is normally a moment full of excitement — but at one Florida high school, some students were left in shock after seeing that their yearbook portraits had been edited.
When Bartram Trail High School freshman Riley O’Keefe saw her yearbook photo, she noticed a black bar was added to cover more of her breasts.
“I couldn’t believe that they printed the yearbook looking like that,” O’Keefe, 15, told CNN. “And then I started to flip through the yearbook and saw more and more girls with their chest edited.”
“I know she’s worn (the outfit) in school hundreds of times because it’s like her go-to outfit,” her mother Stephanie Fabre told CNN on Monday, saying she believes the outfit had met the district’s dress code. The dress code states that girls’ tops “must cover the entire shoulder and they must be modest and not revealing or distracting.”
There were 80 photos of female students that were altered in the yearbook this year, the district’s Chief of Community Relations Christina Langston told CNN on Monday.
The high school’s website has a disclaimer saying that if student portraits in the yearbook did not match the district’s student code of conduct, they may be “digitally adjusted.”
The superintendent said there was an insufficient review before the school decided to edit some of the students’ images. He called the staff member involved an “outstanding educator” and said there will be changes in how the content is considered in yearbooks to come.
The student dress code prohibits clothing that is “immodest, revealing, or distracting,” according to the district’s code of conduct. However, each school’s principal has the “final authority” over whether a student’s dress is appropriate, the code says.
The anger over the edited yearbook photos is part of a larger issue, said Fabre. It’s the district’s dress code that needs to be revisited for its inequity with how it treats what girls wear, as opposed to boys, the student’s parent said.
CRISPR diagnostics | Science
FDA-authorized CRISPR-dx tests are currently only for use in centralized labs, because the most common CRISPR detection protocols require fluid handling steps and two different incubations, precluding their immediate use at the point of care. Single-step formulations have been developed to overcome this limitation, and these "one-pot" versions of CRISPR-dx are simple to run, operate at a single temperature, and run without complex equipment, producing either fluorescence or lateral flow readouts. The programmability of CRISPR makes new diagnostic tests easier to develop, and within months of the release of the SARS-CoV-2 genome, many COVID-19–specific CRISPR tests were reported and distributed around the world.
In just 5 years, the CRISPR-dx field has rapidly expanded, growing from a set of peculiar molecular biology discoveries to multiple FDA-authorized COVID-19 tests and spanning four of the six major subtypes of CRISPR systems. Despite the tremendous promise of CRISPR-dx, substantial challenges remain to adapting these technologies for point-of-care and at-home settings. Simplification of the chemistries to operate as a single reaction in a matter of minutes would be revolutionary, especially if the reaction could be run at room temperature without any complex or expensive equipment. These improvements to CRISPR-dx assays can be achieved by identification or engineering of additional Cas enzymes with lower-temperature requirements, higher sensitivity, or faster kinetics, enabling rapid and simple amplification-free detection with single-molecule sensitivity.
Coronavirus 'optimized for transmission between humans' in way never before seen:
Former State Department contractor David Asher reacts to the latest review of the Wuhan lab leak theory by the Biden administration.
"It came out of nowhere, and it was optimized for transmission between humans in a way that no bat-borne coronavirus ever had been. So what's up? A pangolin gets mated with a bat gets mated with a furin cleavage site of a human being? How does that happen? It's not probable. In fact, it was statistically 1 in 13 million to 1 in 13 billion, when I talked to our bio-statisticians," he told " The Story ."
The virus' true origins remain unknown, but the possibility it accidentally leaked from a Wuhan lab known for its risky coronavirus experiments has gained increasing credence in recent months. The Wuhan Institute of Virology has conducted so-called "gain of function" research, which involves modifying a virus to make it more infectious among humans.
FORMER STATE DEPARTMENT OFFICIAL: PROBE INTO COVID ORIGINS FOUND ALMOST NO EVIDENCE SUPPORTING NATURAL ORIGIN
Asher helped draft the Trump State Department memo released Jan. 15 that said the Wuhan Institute of Virology was not a merely civilian institution and had collaborated on secret projects with the Chinese military since 2017.
"We had very definitive information, only a bit of which we put out publicly, that showed they were engaged in this classified, secret effort to develop these super viruses and that it appeared it had spilled out of that laboratory," he said. "That was the only plausible information that we were presented."
Asher said the National Institutes of Health and CDC wouldn't provide proof that the virus originated from nature, calling it a "joke."
Asher's investigation came out of the State Department's arms control and verification (AVC) bureau and initially launched at the request of former Trump Secretary of State Mike Pompeo before ending this year.
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Researchers identify a novel SARS-CoV-2 variant (with the V1230L mutation) in West Bengal,
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Several variants of concern have emerged in different parts of the globe, like the B.1.1.7 in the United Kingdom, the B.1.351 in South Africa, and the P.1 strain in Brazil. Two new variants have recently been reported from India, the B.1.617 and B.1.618 strains, characterized by several mutations in the spike protein.
The frequency of the B.1.617 variant in the population increased significantly beginning February 2021. This variant was reported to be seen in the United Kingdom, United States, and Singapore in later February 2021, spreading to more than 60 countries by May 2021.
In a study published on the medRxiv * preprint server, researchers report the emergence of a new strain of the SARS-CoV-2 virus in the state of West Bengal in India.
The researchers obtained full genome sequences of 2,000 SARS-CoV-2 strains collected between January and March 2021 in India from the GISAID database. They performed a mutational analysis, obtained the amino acid sequence, and compared the sequences with the Wuhan strain.
After further analysis, the team found 70 strains with a new set of 11 mutations. There were three mutations in the non-structural proteins, three spike protein mutations, and two nucleocapsid protein mutations.
Sixteen of the sequences had the E484K mutation along with D614G, P681H, and V1230L mutations. These mutations fall in the GR clade, which has four other mutations that were also present in these strains. Further analysis of these variants revealed that these strains form a new cluster within the GR clade.
P681 is present next to the furin cleavage site, where proteolytic cleavage occurs between arginine (R685) and serine (S686). This site is at the junction between the S1 subunit, responsible for binding to the angiotensin-converting enzyme 2 (ACE2) receptor, and the S2 subunit, responsible for fusion between the cell membrane and the virus. Hence, the presence of a mutation at this site could affect S1/S2 cleavage and the infectiousness of the virus.
The P681H mutation has been seen before in the variant from the United Kingdom and is believed to increase spike protein cleavage, but does not seem to affect viral entry or its spread. The V1230L mutation has not been seen before and is located in the transmembrane region of the S2 subunit, which anchors the spike protein to the virus envelope. The substitution of valine with leucine could increase the binding of the spike protein to the viral envelope.
Demi Lovato pays tribute to Elton John in first performance since coming out as non-binary |
On Thursday, Demi Lovato made her first public appearance since announcing they non-binary to fans and have changed their pronouns to them/they.
The pop singer opted for an androgynous look, as they arrived in a blue velvet suit at the the iHeartRadio Music Awards, held at the Dolby Theater in Los Angeles before heading to dinner at Craig's with their inspiration Elton John.
Earlier in the night, Demi sported an ensemble that displayed their cleavage with a plunging neckline, and the singer wore a similarly chic embroidered suit when later performing a tribute to Elton on-stage.
Out and about: On Thursday, Demi Lovato made her first public appearance since announcing they non-binary to fans and have changed their pronouns to them/they, as she attended the iHeartRadio Awards before enjoying dinner with their inspiration Elton John
Their ensemble at the awards with the Elton-inspired ensemble including big bedazzled sunglasses as she belted out his hits on stage.
Also paying tribute to Elton - who arrived at the ceremony with his partner David Furnish - were performers H.E.R. and Brandi Carlile.
Demi and Elton formed a friendship through their shared sobriety and when his biopic Rocket Man came out, Demi lauded the star.
She wrote on Instagram: 'Elton, you are such an icon and I'm honored to know you, not because of all you've accomplished or created...
In stitches! Earlier in the night, she had honoured the music legend - both with a vocal tribute and by donning an ensemble inspired by his kooky style
Physics - How a Virus Rolls Itself Across a Cell Surface
Before an influenza virus invades a cell in the airway, it stretches into a filamentary structure that rolls across the cell’s surface like a pencil on a desktop. Two theorists now propose a new mechanism for the rolling [ 1 ]. They show that two proteins on the viral surface act together to propel the motion through interactions with the cell membrane. Understanding this process might point to ways of preventing infection. However, some experts will not be convinced without more detailed modeling.
The tight grip is supplied by a “spike protein” called hemagglutinin (HA) that protrudes from the viral surface and binds to a chemical group called sialic acid on the surface of a cell membrane. The bond is temporary, as each HA continually disconnects and reconnects to a new sialic acid. Even so, with many of these bonds existing at any given time, they would hold the virus in place. But there is a second spike protein called neuraminidase (NA) that cuts off sialic acid groups in the contact region, gradually reducing the number of binding sites available for HA. Experiments show that without HA, the virus can’t attach; without NA, it can’t move [ 2 ].
But how do HA and NA act together to produce concerted rolling motion, rather than, say, a disorganized succession of binding and cleavage events? To explain that, physicists Falko Ziebert of Heidelberg University in Germany and Igor Kulić of the Charles Sadron Institute in Strasbourg, France, have now devised a model that adapts ideas developed for other biological “motors” that produce molecular-scale motion. They conclude that the virus rolling operates in a way not previously identified in biological systems.
Using both model calculations and computer simulations, the two researchers find that this process can produce persistent rolling motion. Once the symmetry of the system is broken, pulling the virus in one specific direction, the interplay of HA and NA creates a torque that keeps the virus rolling. Occasionally, the virus particles can, through chance fluctuations in the binding and cleavage events, reverse direction and set off in a new direction.
“The really surprising thing is that the rolling is almost independent of the system parameters,” such as the NA cleavage rate, says Kulić—it seems to be virtually inevitable as long as the virus remains stuck to the membrane. The researchers say that their proposed mechanism is probably similar to that operating in a recently reported artificial system involving silica nanoparticles coated with DNA that roll over a surface covered with RNA when a bond-cutting enzyme is present [ 3 ].
“If this result is correct, it would open important vistas for constructing very simple enzyme-powered molecular machines,” says biophysicist Dean Astumian of the University of Maine, a specialist in molecular and nanoscale motors. However, in the absence of a more detailed model, he is skeptical that the mechanism explains how the virus can break the symmetry and begin rolling steadily in one direction. Kulić agrees that the virus is frozen in place at first but says that that state doesn’t last.
Virologist Erik de Vries of Utrecht University in the Netherlands says that “any physical model or theory to dissect and quantify the rolling behavior of IVA is highly valuable.” But he says that more data on the reaction rates of HA and NA are needed to properly test the model.
Philip Ball is a freelance science writer in London. His latest book is How To Grow a Human (University of Chicago Press, 2019).
Researchers use nonequilibrium statistical physics methods to guide the design of vaccines that are effective against many strains of a virus, a holy grail of immunology. Read More »
Noncanonical crRNAs derived from host transcripts enable multiplexable RNA detection by Cas9 |
( A ) Coimmunoprecipitation and sequencing RNAs bound to Cas9-3xFLAG from C. jejuni CG8421 using RIP-seq. ( B ) Western blot analysis of samples from C. jejuni strains with Cas9-3xFLAG or untagged WT control before and after immunoprecipitation. ( C ) Size range of the cellular RNA fragments identified through RIP-seq. Colors indicate the class of RNA. ( D ) Two motifs extracted by MEME from the enriched RNA fragments and their predicted interaction with crRNA2 or the tracrRNA. E -value = 8.8 × 10 −22 (motif 1), 8.9 × 10 −14 (motif 2). ( E ) Distribution of RNA lengths centered around motif #2. ( F ) Predicted binding affinity ( K a ) between the tracrRNA anti-repeat and each enriched RNA fragment. Orange indicates the presence of motif #2. ( G ) Predicted fliF mRNA:tracrRNA duplex and mapped reads from differential RNA-seq (top) or RIP-seq (bottom) performed in CG8421. ( H ) Northern blot analysis for fliF RNAs from CG8421 WT and cas9 -deletion strains.
These crRNA-like RNAs raised the question of whether these same RNAs were present in our prior RIP-seq analysis with strain NCTC11168 ( 9 ). We found that 7 of the 96 enriched fragments were predicted to bind the tracrRNA anti-repeat more tightly than at least one crRNA (fig. S3, A and B). Two of these RNAs (derived from fliF and dctA mRNAs) matched those found in CG8421 ( Fig. 1G and figs. S2D and S3C). The RNA fragment derived from the fliF mRNA could be detected by Northern blot in both total RNA and RIP-seq samples yet disappeared following deletion of cas9 ( Fig. 1H and fig. S4, A and B). The dctA RNA fragment was only weakly detected in one strain (fig. S4). Although cas9 deletion did not significantly perturb FliF protein concentrations in vivo under standard growth conditions (fig. S5), deleting the CRISPR array in NCTC11168 increased levels of the fliF RNA fragment (fig. S4C). Finally, the fliF RNA fragment was a processing product, as confirmed with Terminator exonuclease treatment ( Fig. 1G ). These crRNA-like RNAs thus are also present in C. jejuni NCTC11168 and likely exist in other C. jejuni strains on account of the shared tracrRNA binding site in fliF (fig. S6).
Cas9 binding, predicted tracrRNA pairing, and the length distribution of many of these enriched RNA fragments suggested that the tracrRNA pairs with endogenous RNAs, resulting in "noncanonical" crRNAs (ncrRNAs) ( Fig. 2A and fig. S2A). The ncrRNAs therefore would be expected to direct Cas9 to complementary DNA targets flanked by a protospacer-adjacent motif (PAM), similar to a canonical crRNA ( 12 ). As none of the genes giving rise to the detected ncrRNAs has a correctly placed PAM, the ncrRNAs are not expected to direct Cas9 to cleave their originating genomic site (table S1).
( A ) General process for ncrRNA generation. ( B ) Applying the TXTL assay to characterize putative ncrRNAs. ( C ) DNA targeting through the fliF ncrRNA in TXTL. Lines and shaded regions indicate the mean and standard deviation from four separately mixed replicates. NT, nontargeting. ( D ) Systematic evaluation of mutating the repeat:anti-repeat duplex for CjeCas9 with TXTL. Endpoint GFP levels are shown. Mutations and extensions to the fliF sgRNA-crRNA repeat are indicated in red. See table S1 for sequences. ( E ) DNA targeting by selected ncrRNAs predicted in TXTL. Check marks indicate use of the construct above the line. mRNA(mut): mRNA encoding the ncrRNA with point mutations in the predicted "seed" region of the guide. tracrRNA(scr): tracrRNA with the anti-repeat sequence scrambled. Values in (D) and (E) represent the mean and standard deviation from four separately mixed replicates. ** p < 0.001. n.s., not significant.
The reduced performance of the fliF mRNA in TXTL could be due to how an ncrRNA deviates from a standard crRNA. These deviations include the crRNA repeat sequence, the secondary structure of the duplex formed with the tracrRNA anti-repeat, and 5′ or 3′ extensions to the repeat that do not undergo efficient processing. To evaluate these deviations, we systematically mutated or extended the standard crRNA, either as a single guide RNA (sgRNA) to ensure duplex formation or as a crRNA:tracrRNA pair, and evaluated GFP silencing in TXTL ( Fig. 2D and table S1). CjeCas9 could accommodate some mutations within the region of the repeat:anti-repeat duplex in the sgRNA implicated in nuclease binding ( 16 ). The more disruptive mutations spanned more nts, were closer to the 5′ end of the repeat, or resulted in a bulge in the tracrRNA (e.g., s3, s6, s7, s9, s12, s18 in Fig. 2D , left). Observed differences in GFP silencing do not appear to arise from variable sgRNA levels (fig. S7B). Extending the sgRNA-crRNA ends or mutating the region cleaved by RNase III within the crRNA had minimal impact on GFP silencing ( Fig. 2D , right). Overall, the tracrRNA can tolerate deviations from a standard crRNA as long as pairing through the 3′ end of the tracrRNA anti-repeat is maintained.
We applied insights from our mutational analyses to prioritize putative ncrRNAs from C. jejuni CG8421 for functional tests in TXTL. In total, we identified eight RNA fragments predicted to base pair extensively with the 3′ end of the tracrRNA anti-repeat ( Fig. 2E and fig. S8). We then assessed GFP silencing by expressing up to 350 nts upstream and downstream of each associated ncrRNA-encoding gene with tracrRNA, CjeCas9, and the GFP reporter harboring each cognate DNA target. Of the eight tested RNAs, three (from rseP , nuoL , and dctA ) yielded a >twofold reduction in GFP reporter levels compared with a nontargeting crRNA control ( p < 0.001). Furthermore, targeting was directed specifically through the predicted ncrRNA, as mutating the "seed" region of the putative ncrRNA ( 17 ), scrambling the tracrRNA anti-repeat, or replacing CjeCas9 with the orthogonal FnCas12a nuclease fully relieved GFP repression ( Fig. 2E ). Multiple factors, such as mRNA folding or accessibility during translation, may explain why the other five ncrRNAs did not exhibit targeting activity in TXTL, as a linear-regression model built around the sgRNA mutants had limited ability to predict the targeting activity of these ncrRNAs (supplementary text S1). The current lack of predictability parallels guide design for RNA-targeting Cas13a nucleases, which only became predictable with extensive datasets and machine learning ( 18 ).
Beyond TXTL, we assessed ncrRNA function as part of DNA targeting in C. jejuni CG8421 and in Escherichia coli . For CG8421, transformation interference assays did not yield any significant DNA targeting directed by ncrRNAs derived from the rseP , dctA , and nuoL mRNAs (fig. S9A), likely due to low ncrRNA abundance compared with the strain's crRNAs under the examined growth conditions. For E. coli , overexpressing the dctA mRNA, CjeCas9, and the tracrRNA led to moderate (15.5-fold) clearance of a transformed plasmid with the putative dctA ncrRNA target ( p = 0.0036), but not when the tracrRNA anti-repeat was scrambled (1.6-fold) ( p = 0.068) (fig. S9B). We therefore conclude that ncrRNAs derived from mRNAs can elicit DNA targeting in both in vivo and cell-free systems.
The conversion of a cellular RNA into an ncrRNA was based on sequences bearing complementarity to the tracrRNA anti-repeat, analogous to natural crRNA biogenesis (fig. S2A). What if the tracrRNA anti-repeat sequence could be changed to hybridize to other RNAs while maintaining the appropriate structure for Cas9 recognition? If so, then the resulting reprogrammed tracrRNA (Rptr, pronounced "raptor") could specifically derive an ncrRNA from a cellular RNA. The resulting ncrRNAs can then guide Cas9 to matching DNA targets ( Fig. 3A ). Although tracrRNA engineering has rarely been explored outside of sgRNAs or crRNA:tracrRNA duplexes ( 19 ), multiple studies have shown that the repeat:anti-repeat duplex of the sgRNA for the Streptococcus pyogenes Cas9 (SpyCas9) can be extensively modified as long as the secondary structure is maintained ( 20 , 21 ).
( A ) Design of reprogrammed tracrRNAs (Rptrs) utilized by CjeCas9. W = A or T. PAM, yellow circle. ( B ) Toleration of mutations to the RNA duplex of an sgRNA that preserve secondary structure in TXTL. ( C ) Enhancing DNA targeting by less functional or non-functional ncrRNAs by converting the tracrRNA into a Rptr in TXTL. ( D ) Sequence-specific DNA targeting in TXTL using Rptrs compatible with three different Cas9 nucleases. Values in (B) to (D) represent the mean and standard deviation from four replicates in TXTL. * p < 0.01; ** p < 0.001. n.s., not significant.
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