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SNP-Chip: CRISPR-Based Transistor Detects Single Point Mutations

The CRISPR-based "SNP-Chip" is a graphene field-effect transistor (gFET) that enables amplification-free electronic detection of point mutations. A new, proof of concept study illustrates that the SNP-Chip has the ability to detect point mutations in…

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Long before the COVID-19 pandemic shined a spotlight on CRISPR-based diagnostics, Kiana Aran, PhD, was developing the CRISPR-Chip. It was, according to Aran, the first transistor that uses CRISPR to search the genome for potential mutations. In addition, the graphene field-effect transistor (gFET) did not require amplification or sequencing of the nucleic acid.

Now, two years after its introduction, a group headed by Aran, co-founder and CSO at Cardea Bio, and assistant professor at the Keck Graduate Institute of Applied Life Sciences, is introducing an improved CRISPR-based gFET system, named SNP-Chip.

Kiana Aran, PhD

The two advances in the SNP-chip compared to its predecessor are 1) improved electronics that allow for more, higher quality, measurements and 2) the incorporation of a novel Cas enzyme (a Cas9 ortholog). The ability of the SNP-chip to identify single nucleotide polymorphisms (SNPs) was illustrated in two genetic diseases: amyotrophic lateral sclerosis (ALS) and sickle cell disease (SCD).

The work is published online today in Nature Biomedical Engineering in a paper titled, “Discrimination of single-point mutations in unamplified genomic DNA via Cas9 immobilized on a graphene field-effect transistor.”

This is a nice proof of concept study, asserted Rodolphe Barrangou, PhD, professor in the department of food, bioprocessing, and nutrition sciences at North Carolina State University, and editor-in-chief of The CRISPR Journal, that showcases the potential of the previously developed method, with convincing SCD and ALS targets. “Being able to detect SNPs,” Barrangou continued, “especially without the need for amplification, is noteworthy and opens new avenues for CRISPR-based diagnostics.”

The authors noted that the SNP-Chip is not the first new technology that has been developed to perform SNP genotyping without sequencing. Some technologies in this space have removed the need for expensive optical equipment while others have cut out the need for amplification. But no previous methods have achieved both.

Michael Heltzen, CEO of Cardea Bio, told GEN that he believes the SNP-Chip is, “the biggest genetic breakthrough since PCR was invented in 1983.” While Barrangou asserts that the SNP-Chip “could be a game-changer for precision diagnostics,” he added that it is “not on the same revolutionary scale as PCR.”

Detecting disease-causing SNPs in DNA

For the past two years, Aran told GEN, her team has been working toward point mutation detection in the SNP-Chip. This goal came largely from listening to what their customers wanted.

The current work illustrates the detection of SNPs by the SNP-Chip in two human disease models. When the team tested the SNP-Chip on genomic samples isolated from three patients with SCD and three healthy individuals, the SNP-Chip could differentiate between the two. In ALS, the SNP-Chip could discriminate between genomic DNA extracted from human induced pluripotent stem cells (hiPSCs) from a healthy individual and those from an individual with familial ALS (carrying the H44R mutation in the superoxide dismutase type 1 (SOD1) gene). In the SCD model, the SNP-Chip was also able to detect heterozygosity of SNPs without DNA amplification.

CRISPR SNP-Chip can detect and quantify single point mutation directly from unamplified whole genomic DNA samples without laborious sample preparation or optical instruments. [Cardea Bio]
Aran stressed that success in detecting mutations associated with these two diseases is just the beginning. Using a new guide RNA, which would reconfigure the CRISPR-element employed within SNP-Chip, is all that is needed to target different mutations.

A Cas enzyme from a Cas expert      

One of Anan’s co-authors is Virginijus Šikšnys, the Lithuanian biochemist who has been in the CRISPR field almost before it existed. Despite his 2012 paper bearing a later publication date than that of the infamous Science paper co-authored by Jennifer Doudna, Emmanuelle Charpentier, and colleagues, his related discoveries were made no later than theirs. (He shared the 2018 Kavli Prize with Doudna and Charpentier.)

Last year, Šikšnys told GEN, with regards to non-genome editing applications, “programmable CRISPR-Cas-based detection technologies will expand because of the ease of changing recognition sequence and their specificity. On this front, we will likely see even more CRISPR-Cas applications in the diagnostics space.” By teaming up with Cardea Bio, Šikšnys is now playing a central role in making this happen.

The first CRISPR-Chip utilized the sequence-specific gene-targeting properties of a deactivated Cas9 enzyme. The deactivated Cas yielded a big signal, according to Aran, but Cardea found the commercial accessibility to be a hurdle. So, Cardea turned to Šikšnys and his company, CasZyme, for alternative Cas orthologs. The Cas9 orthologue that is used in the SNP-Chip comes from Mycoplasma gallisepticum CA06 strain (MgaCas9).

Šikšnys noted that merging a diversity of CRISPR-Cas biology with electronics via Cardea transistors opens up “a whole new range of possibilities for diagnostic and research applications.” He added that “using the Cas9 orthologue for SNP detection is just the tip of the iceberg of opportunities.”

Endless possibilities

The most immediate application where Aran envisions SNP-Chip making an impact is in monitoring CRISPR-based gene editing quality control processes. The SNP-Chip can ascertain the efficiency of the editing process by measuring how many cells were edited. There will likely be more applications in the SNP-Chip’s future, as it can theoretically detect SNPs in any genetic material—from agricultural and environmental monitoring to human disease.

The CRISPR-based SNP-Chip is dependent on protospacer adjacent motif (PAM), which creates a limitation when targeting a SNP. If a PAM is not near the target site, it will be more difficult to detect because Cas9 cannot gain access. Šikšnys previously told GEN that even the most versatile SpyCas9 variant cannot access approximately 75% of the single-base mutations that are associated with human disease, because there is no appropriately located PAM. Barrangou added that it is important to note “the continued need to encompass orthogonal Cas9 effectors so PAM-related limitations are overcome using biodiversity.”

Could the SNP-Chip detect variants in emerging (and evolving) viruses like SARS-CoV-2? At the moment, one chip is limited to detecting only one point mutation. In order to detect more point mutations, Aran explained, you would need more than one chip. But the team is working on this improvement by creating a 4-plex chip with hopes of a 16-plex chip.

Today, SNP-Chip experiments are done in the San Diego office of Cardea Bio. But the goal, according to Aran, is to sell disposable chips so that anyone can use them, anywhere.

The late Kary Mullis, PhD, inventor of PCR, once said that “science consistently produces a new crop of miraculous truths and dazzling devices every year.” The role that the SNP-Chip will play in the future of CRISPR diagnostics, and other applications, remains to be seen. Nevertheless, it is an early contender in a new era of amplification-free SNP detection.

The post SNP-Chip: CRISPR-Based Transistor Detects Single Point Mutations appeared first on GEN - Genetic Engineering and Biotechnology News.

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Survey Shows Declining Concerns Among Americans About COVID-19

Survey Shows Declining Concerns Among Americans About COVID-19

A new survey reveals that only 20% of Americans view covid-19 as "a major threat"…

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4 hours ago

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March 11, 2024

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Survey Shows Declining Concerns Among Americans About COVID-19

A new survey reveals that only 20% of Americans view covid-19 as "a major threat" to the health of the US population - a sharp decline from a high of 67% in July 2020.

(SARMDY/Shutterstock)

What's more, the Pew Research Center survey conducted from Feb. 7 to Feb. 11 showed that just 10% of Americans are concerned that they will  catch the disease and require hospitalization.

"This data represents a low ebb of public concern about the virus that reached its height in the summer and fall of 2020, when as many as two-thirds of Americans viewed COVID-19 as a major threat to public health," reads the report, which was published March 7.

According to the survey, half of the participants understand the significance of researchers and healthcare providers in understanding and treating long COVID - however 27% of participants consider this issue less important, while 22% of Americans are unaware of long COVID.

What's more, while Democrats were far more worried than Republicans in the past, that gap has narrowed significantly.

"In the pandemic’s first year, Democrats were routinely about 40 points more likely than Republicans to view the coronavirus as a major threat to the health of the U.S. population. This gap has waned as overall levels of concern have fallen," reads the report.

More via the Epoch Times;

The survey found that three in ten Democrats under 50 have received an updated COVID-19 vaccine, compared with 66 percent of Democrats ages 65 and older.

Moreover, 66 percent of Democrats ages 65 and older have received the updated COVID-19 vaccine, while only 24 percent of Republicans ages 65 and older have done so.

“This 42-point partisan gap is much wider now than at other points since the start of the outbreak. For instance, in August 2021, 93 percent of older Democrats and 78 percent of older Republicans said they had received all the shots needed to be fully vaccinated (a 15-point gap),” it noted.

COVID-19 No Longer an Emergency

The U.S. Centers for Disease Control and Prevention (CDC) recently issued its updated recommendations for the virus, which no longer require people to stay home for five days after testing positive for COVID-19.

The updated guidance recommends that people who contracted a respiratory virus stay home, and they can resume normal activities when their symptoms improve overall and their fever subsides for 24 hours without medication.

“We still must use the commonsense solutions we know work to protect ourselves and others from serious illness from respiratory viruses, this includes vaccination, treatment, and staying home when we get sick,” CDC director Dr. Mandy Cohen said in a statement.

The CDC said that while the virus remains a threat, it is now less likely to cause severe illness because of widespread immunity and improved tools to prevent and treat the disease.

“Importantly, states and countries that have already adjusted recommended isolation times have not seen increased hospitalizations or deaths related to COVID-19,” it stated.

The federal government suspended its free at-home COVID-19 test program on March 8, according to a website set up by the government, following a decrease in COVID-19-related hospitalizations.

According to the CDC, hospitalization rates for COVID-19 and influenza diseases remain “elevated” but are decreasing in some parts of the United States.

Tyler Durden Sun, 03/10/2024 - 22:45

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Rand Paul Teases Senate GOP Leader Run – Musk Says “I Would Support”

Rand Paul Teases Senate GOP Leader Run – Musk Says "I Would Support"

Republican Kentucky Senator Rand Paul on Friday hinted that he may jump…

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7 hours ago

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March 10, 2024

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Rand Paul Teases Senate GOP Leader Run - Musk Says "I Would Support"

Republican Kentucky Senator Rand Paul on Friday hinted that he may jump into the race to become the next Senate GOP leader, and Elon Musk was quick to support the idea. Republicans must find a successor for periodically malfunctioning Mitch McConnell, who recently announced he'll step down in November, though intending to keep his Senate seat until his term ends in January 2027, when he'd be within weeks of turning 86. 

So far, the announced field consists of two quintessential establishment types: John Cornyn of Texas and John Thune of South Dakota. While John Barrasso's name had been thrown around as one of "The Three Johns" considered top contenders, the Wyoming senator on Tuesday said he'll instead seek the number two slot as party whip. 

Paul used X to tease his potential bid for the position which -- if the GOP takes back the upper chamber in November -- could graduate from Minority Leader to Majority Leader. He started by telling his 5.1 million followers he'd had lots of people asking him about his interest in running...

...then followed up with a poll in which he predictably annihilated Cornyn and Thune, taking a 96% share as of Friday night, with the other two below 2% each. 

Elon Musk was quick to back the idea of Paul as GOP leader, while daring Cornyn and Thune to follow Paul's lead by throwing their names out for consideration by the Twitter-verse X-verse. 

Paul has been a stalwart opponent of security-state mass surveillance, foreign interventionism -- to include shoveling billions of dollars into the proxy war in Ukraine -- and out-of-control spending in general. He demonstrated the latter passion on the Senate floor this week as he ridiculed the latest kick-the-can spending package:   

In February, Paul used Senate rules to force his colleagues into a grueling Super Bowl weekend of votes, as he worked to derail a $95 billion foreign aid bill. "I think we should stay here as long as it takes,” said Paul. “If it takes a week or a month, I’ll force them to stay here to discuss why they think the border of Ukraine is more important than the US border.”

Don't expect a Majority Leader Paul to ditch the filibuster -- he's been a hardy user of the legislative delay tactic. In 2013, he spoke for 13 hours to fight the nomination of John Brennan as CIA director. In 2015, he orated for 10-and-a-half-hours to oppose extension of the Patriot Act. 

Rand Paul amid his 10 1/2 hour filibuster in 2015

Among the general public, Paul is probably best known as Capitol Hill's chief tormentor of Dr. Anthony Fauci, who was director of the National Institute of Allergy and Infectious Disease during the Covid-19 pandemic. Paul says the evidence indicates the virus emerged from China's Wuhan Institute of Virology. He's accused Fauci and other members of the US government public health apparatus of evading questions about their funding of the Chinese lab's "gain of function" research, which takes natural viruses and morphs them into something more dangerous. Paul has pointedly said that Fauci committed perjury in congressional hearings and that he belongs in jail "without question."   

Musk is neither the only nor the first noteworthy figure to back Paul for party leader. Just hours after McConnell announced his upcoming step-down from leadership, independent 2024 presidential candidate Robert F. Kennedy, Jr voiced his support: 

In a testament to the extent to which the establishment recoils at the libertarian-minded Paul, mainstream media outlets -- which have been quick to report on other developments in the majority leader race -- pretended not to notice that Paul had signaled his interest in the job. More than 24 hours after Paul's test-the-waters tweet-fest began, not a single major outlet had brought it to the attention of their audience. 

That may be his strongest endorsement yet. 

Tyler Durden Sun, 03/10/2024 - 20:25

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The Great Replacement Loophole: Illegal Immigrants Score 5-Year Work Benefit While “Waiting” For Deporation, Asylum

The Great Replacement Loophole: Illegal Immigrants Score 5-Year Work Benefit While "Waiting" For Deporation, Asylum

Over the past several…

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March 10, 2024

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The Great Replacement Loophole: Illegal Immigrants Score 5-Year Work Benefit While "Waiting" For Deporation, Asylum

Over the past several months we've pointed out that there has  been zero job creation for native-born workers since the summer of 2018...

... and that since Joe Biden was sworn into office, most of the post-pandemic job gains the administration continuously brags about have gone foreign-born (read immigrants, mostly illegal ones) workers.

And while the left might find this data almost as verboten as FBI crime statistics - as it directly supports the so-called "great replacement theory" we're not supposed to discuss - it also coincides with record numbers of illegal crossings into the United States under Biden.

In short, the Biden administration opened the floodgates, 10 million illegal immigrants poured into the country, and most of the post-pandemic "jobs recovery" went to foreign-born workers, of which illegal immigrants represent the largest chunk.

Asylum seekers from Venezuela await work permits on June 28, 2023 (via the Chicago Tribune)

'But Tyler, illegal immigrants can't possibly work in the United States whilst awaiting their asylum hearings,' one might hear from the peanut gallery. On the contrary: ever since Biden reversed a key aspect of Trump's labor policies, all illegal immigrants - even those awaiting deportation proceedings - have been given carte blanche to work while awaiting said proceedings for up to five years...

... something which even Elon Musk was shocked to learn.

Which leads us to another question: recall that the primary concern for the Biden admin for much of 2022 and 2023 was soaring prices, i.e., relentless inflation in general, and rising wages in particular, which in turn prompted even Goldman to admit two years ago that the diabolical wage-price spiral had been unleashed in the US (diabolical, because nothing absent a major economic shock, read recession or depression, can short-circuit it once it is in place).

Well, there is one other thing that can break the wage-price spiral loop: a flood of ultra-cheap illegal immigrant workers. But don't take our word for it: here is Fed Chair Jerome Powell himself during his February 60 Minutes interview:

PELLEY: Why was immigration important?

POWELL: Because, you know, immigrants come in, and they tend to work at a rate that is at or above that for non-immigrants. Immigrants who come to the country tend to be in the workforce at a slightly higher level than native Americans do. But that's largely because of the age difference. They tend to skew younger.

PELLEY: Why is immigration so important to the economy?

POWELL: Well, first of all, immigration policy is not the Fed's job. The immigration policy of the United States is really important and really much under discussion right now, and that's none of our business. We don't set immigration policy. We don't comment on it.

I will say, over time, though, the U.S. economy has benefited from immigration. And, frankly, just in the last, year a big part of the story of the labor market coming back into better balance is immigration returning to levels that were more typical of the pre-pandemic era.

PELLEY: The country needed the workers.

POWELL: It did. And so, that's what's been happening.

Translation: Immigrants work hard, and Americans are lazy. But much more importantly, since illegal immigrants will work for any pay, and since Biden's Department of Homeland Security, via its Citizenship and Immigration Services Agency, has made it so illegal immigrants can work in the US perfectly legally for up to 5 years (if not more), one can argue that the flood of illegals through the southern border has been the primary reason why inflation - or rather mostly wage inflation, that all too critical component of the wage-price spiral  - has moderated in in the past year, when the US labor market suddenly found itself flooded with millions of perfectly eligible workers, who just also happen to be illegal immigrants and thus have zero wage bargaining options.

None of this is to suggest that the relentless flood of immigrants into the US is not also driven by voting and census concerns - something Elon Musk has been pounding the table on in recent weeks, and has gone so far to call it "the biggest corruption of American democracy in the 21st century", but in retrospect, one can also argue that the only modest success the Biden admin has had in the past year - namely bringing inflation down from a torrid 9% annual rate to "only" 3% - has also been due to the millions of illegals he's imported into the country.

We would be remiss if we didn't also note that this so often carries catastrophic short-term consequences for the social fabric of the country (the Laken Riley fiasco being only the latest example), not to mention the far more dire long-term consequences for the future of the US - chief among them the trillions of dollars in debt the US will need to incur to pay for all those new illegal immigrants Democrat voters and low-paid workers. This is on top of the labor revolution that will kick in once AI leads to mass layoffs among high-paying, white-collar jobs, after which all those newly laid off native-born workers hoping to trade down to lower paying (if available) jobs will discover that hardened criminals from Honduras or Guatemala have already taken them, all thanks to Joe Biden.

Tyler Durden Sun, 03/10/2024 - 19:15

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