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How to design a permanent vaccine to cholera using CRISPR?


I know that Vibrio Cholerae infects the body through the GM1 ganglioside. So, would it be possible to engineer a CRISPR gene editing tool to prevent Vibrio Cholerae from getting into our cells?

Specifically, would it be possible to insert a piece of DNA into our genes that allow the synthesis of cholera toxin-suspressing protein shown here (Pubmed link).

The reason I ask this question is that there are obvious benefits to being immune to cholera from birth. While I'm definitely not planning on being the next He Jiankui, I find the prospect of such a vaccine highly intriguing.

Edit: changed link because I posted the wrong link.


So, would it be possible to engineer a CRISPR gene editing tool to prevent Vibrio Cholerae from getting into our cells?

Vibrio Cholerae doesn't get into our cells.

Did you observe that your paper has no in vitro experiments?

It's going to be tough to get a cell to transcribe and translate something so small, and will the peptide work if it needs a leader sequence to be exported out of the cell?

Its much much easier to break a gene in every cell than to add a new gene and have just a small subset of cell types make large quantities of that gene product.


New, reversible CRISPR method can control gene expression while leaving underlying DNA sequence unchanged

A new CRISPR method allows researchers to silence most genes in the human genome without altering the underlying DNA sequence -- and then reverse the changes. Credit: Jennifer Cook-Chrysos/Whitehead Institute

Over the past decade, the CRISPR-Cas9 gene editing system has revolutionized genetic engineering, allowing scientists to make targeted changes to organisms' DNA. While the system could potentially be useful in treating a variety of diseases, CRISPR-Cas9 editing involves cutting DNA strands, leading to permanent changes to the cell's genetic material.

Now, in a paper published online in Cell on April 9, researchers describe a new gene editing technology called CRISPRoff that allows researchers to control gene expression with high specificity while leaving the sequence of the DNA unchanged. Designed by Whitehead Institute Member Jonathan Weissman, University of California San Francisco assistant professor Luke Gilbert, Weissman lab postdoc James Nuñez and collaborators, the method is stable enough to be inherited through hundreds of cell divisions, and is also fully reversible.

"The big story here is we now have a simple tool that can silence the vast majority of genes," says Weissman, who is also a professor of biology at MIT and an investigator with the Howard Hughes Medical Institute. "We can do this for multiple genes at the same time without any DNA damage, with great deal of homogeneity, and in a way that can be reversed. It's a great tool for controlling gene expression."

The project was partially funded by a 2017 grant from the Defense Advanced Research Projects Agency to create a reversible gene editor. "Fast forward four years [from the initial grant], and CRISPRoff finally works as envisioned in a science fiction way," says co-senior author Gilbert. "It's exciting to see it work so well in practice."

Genetic engineering 2.0

The classic CRISPR-Cas9 system uses a DNA-cutting protein called Cas9 found in bacterial immune systems. The system can be targeted to specific genes in human cells using a single guide RNA, where the Cas9 proteins create tiny breaks in the DNA strand. Then the cell's existing repair machinery patches up the holes.

Because these methods alter the underlying DNA sequence, they are permanent. Plus, their reliance on "in-house" cellular repair mechanisms means it is hard to limit the outcome to a single desired change. "As beautiful as CRISPR-Cas9 is, it hands off the repair to natural cellular processes, which are complex and multifaceted," Weissman says. "It's very hard to control the outcomes."

That's where the researchers saw an opportunity for a different kind of gene editor—one that didn't alter the DNA sequences themselves, but changed the way they were read in the cell.

This sort of modification is what scientists call "epigenetic"—genes may be silenced or activated based on chemical changes to the DNA strand. Problems with a cell's epigenetics are responsible for many human diseases such as Fragile X syndrome and various cancers, and can be passed down through generations.

Epigenetic gene silencing often works through methylation—the addition of chemical tags to to certain places in the DNA strand—which causes the DNA to become inaccessible to RNA polymerase, the enzyme which reads the genetic information in the DNA sequence into messenger RNA transcripts, which can ultimately be the blueprints for proteins.

Weissman and collaborators had previously created two other epigenetic editors called CRISPRi and CRISPRa—but both of these came with a caveat. In order for them to work in cells, the cells had to be continually expressing artificial proteins to maintain the changes.

"With this new CRISPRoff technology, you can [express a protein briefly] to write a program that's remembered and carried out indefinitely by the cell," says Gilbert. "It changes the game so now you're basically writing a change that is passed down through cell divisions—in some ways we can learn to create a version 2.0 of CRISPR-Cas9 that is safer and just as effective, and can do all these other things as well."

To build an epigenetic editor that could mimic natural DNA methylation, the researchers created a tiny protein machine that, guided by small RNAs, can tack methyl groups onto specific spots on the strand. These methylated genes are then "silenced," or turned off, hence the name CRISPRoff.

Because the method does not alter the sequence of the DNA strand, the researchers can reverse the silencing effect using enzymes that remove methyl groups, a method they called CRISPRon.

As they tested CRISPRoff in different conditions, the researchers discovered a few interesting features of the new system. For one thing, they could target the method to the vast majority of genes in the human genome—and it worked not just for the genes themselves, but also for other regions of DNA that control gene expression but do not code for proteins. "That was a huge shock even for us, because we thought it was only going to be applicable for a subset of genes," says first author Nuñez.

Also, surprisingly to the researchers, CRISPRoff was even able to silence genes that did not have large methylated regions called CpG islands, which had previously been thought necessary to any DNA methylation mechanism.

"What was thought before this work was that the 30 percent of genes that do not have a CpG island were not controlled by DNA methylation," Gilbert says. "But our work clearly shows that you don't require a CpG island to turn genes off by methylation. That, to me, was a major surprise."

CRISPRoff in research and therapy

To investigate the potential of CRISPRoff for practical applications, the scientists tested the method in induced pluripotent stem cells. These are cells that can turn into countless cell types in the body depending on the cocktail of molecules they are exposed to, and thus are powerful models for studying the development and function of particular cell types.

The researchers chose a gene to silence in the stem cells, and then induced them to turn into nerve cells called neurons. When they looked for the same gene in the neurons, they discovered that it had remained silenced in 90 percent of the cells, revealing that cells retain a memory of epigenetic modifications made by the CRISPRoff system even as they change cell type.

They also selected one gene to use as an example of how CRISPRoff might be applied to therapeutics: the gene that codes for Tau protein, which is implicated in Alzheimer's disease. After testing the method in neurons, they were able to show that using CRISPRoff could be used to turn Tau expression down, although not entirely off. "What we showed is that this is a viable strategy for silencing Tau and preventing that protein from being expressed," Weissman says. "The question is, then, how do you deliver this to an adult? And would it really be enough to impact Alzheimer's? Those are big open questions, especially the latter."

Even if CRISPRoff does not lead to Alzheimer's therapies, there are many other conditions it could potentially be applied to. And while delivery to specific tissues remains a challenge for gene editing technologies such as CRISPRoff, "we showed that you can deliver it transiently as a DNA or as an RNA, the same technology that's the basis of the Moderna and BioNTech coronavirus vaccine," Weissman says.

Weissman, Gilbert, and collaborators are enthusiastic about the potential of CRISPRoff for research as well. "Since we now can sort of silence any part of the genome that we want, it's a great tool for exploring the function of the genome," Weissman says.

Plus, having a reliable system to alter a cell's epigenetics could help researchers learn the mechanisms by which epigenetic modifications are passed down through cell divisions. "I think our tool really allows us to begin to study the mechanism of heritability, especially epigenetic heritability, which is a huge question in the biomedical sciences," Nuñez says.


New genetic method of using CRISPR to eliminate COVID-19 virus genomes in cells

It is predicted the development of a safe and effective vaccine to prevent COVID-19 will take 12 to 18 months, by which time hundreds of thousands to millions of people may have been infected. With a rapidly growing number of cases and deaths around the world, this emerging threat requires a nimble and targeted means of protection.

Could CRISPR be the next virus killer?

To address this global pandemic challenge, we are developing a genetic vaccine that can be used rapidly in healthy and patients to greatly reduce the coronavirus spreading. We developed a safe and effective CRISPR system to precisely target, cut and destroy COVID-19 virus and its genome, which stops coronavirus from infecting the human lung.

We’ve shown that the CRISPR system can reduce 90% of coronavirus load in human cells. It can also protect humans against essentially 90% of all current and emerging coronaviruses. The project is ongoing, and we are working around the clock towards getting an actual product by combing our CRISPR method with an inhaler-based delivery device.

The project will likely to result in a potential therapeutics towards COVID-19, which can help slow down or eliminate the outbreak.


Researchers use CRISPR gene editing to disrupt antibiotic-resistance in bacteria

An encouraging new study from researchers at University of Colorado has shown that disrupting multiple bacterial genes at once is a successful strategy to use against deadly superbugs and emerging antibiotic-resistance in bacteria.

The study was published in Communications Biology on Monday, and widens the scope of using genetic tools to address the growing antibiotic resistance problem by limiting the organism's functioning.

The newly-discovered approach, called Controlled Hindrance of Adaptation of OrganismS (CHAOS) uses the gene-editing tool CRISPR to alter multiple genes in bacterial cells to impair its core functioning abilities.

This cripples some of the central processes in the bacteria — the cell’s defence mechanisms being an important one.

Researchers have developed a combination of “kill switch” genes in Escherichia coli for the approach.

When a single gene in the group is switched off, the bacteria appears to be able to cope, compensate and survive. But on tweaking a combination of 2 or more of these genes, the bacteria got weaker and more sensitive to antibiotic treatments.

Representational Image. Reuters

“We saw that when we tweaked multiple gene expressions at the same time —even genes that would seemingly help the bacteria survive — the bacteria’s fitness dropped dramatically,” Peter Otoupal, lead author of the study said to University Press.

Using this technique doesn’t alter the genome of the bacteria itself, but how the genes are expressed by the cell.

“This method offers tremendous potential to create more effective combinatorial approaches,” Anushree Chatterjee, senior author of the study, said to University Press.

The researchers explain that the method could be further optimized for more efficient disruptions — something the team is pursuing in ongoing research.

“In the past, nobody really considered that it might be possible to slow down evolution,” Otoupal said.

“But like anything else, evolution has rules and we’re starting to learn how to use them to our advantage.”

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2. Origin of CRISPR/Cas System

The CRISPR/Cas system is an important part of adaptive immune mechanisms of bacteria and archaea, which is used to resist foreign genetic plasmids and phage invasion. In 1987, the structure of CRISPR was first reported [15], and then similar structures were identified in different bacteria and archaea species. The CRISPR/Cas system was proposed to be abbreviated as 𠆌RISPR’ in 2002 [16]. In 2005, CRISPR structures and Cas proteins were speculated to have immune defense function and possibly play an important role in protecting genetic factors [17]. Subsequently, the CRISPR/Cas system is proposed to provide resistance against viruses in prokaryotes, which specifically recognizes and binds the DNA of phage through CRISPR RNA (crRNA) and guides Cas proteins to recognize and cleave exogenous DNA through the trans-activating of crRNA (tracrRNA) [18]. According to different Cas protein families and the principles of effector module design, CRISPR/Cas systems have been divided into two classes, with multi-subunit effector complexes in Class 1 and single-protein effector modules in Class 2 [19]. The CRISPR/Cas9 system, belonging to the Class 2 CRISPR/Cas system, is mainly composed of crRNA and Cas9 protein, and only needs a single guide RNA (sgRNA) to precisely cleave the target genes [20,21]. In 2012, the Cas9-crRNA complex was proved to have the ability to cut target DNA in vitro, and the double-stranded breaks (DSBs) occurred at three nucleotides upstream of protospacer adjacent motif (PAM) sequence, realizing it as the first gene-editing tool in a test tube [22]. Subsequently, Charpentier and Doudna’s groups reported the combination of crRNA and tracrRNA into a single sgRNA, which can more efficiently help Cas9 to play its editing role in vitro [23]. In 2013, Zhang’s lab first applied the CRISPR/Cas9 system to perform genome editing in eukaryotic cells [24]. From then on, the new generation of CRISPR gene-editing technology, especially represented by the CRISPR/Cas9 system, has been well developed and widely applied in the field of life sciences, such as to produce gene-edited animal models, gene therapy to treat genetic disease, and animal and plant genetic trait improvement and biological breeding [25,26,27,28,29,30,31]. In 2020, for the epoch-making technological innovation and great contribution to life sciences, two scientists, Charpentier and Doudna, devoted most to the CRISPR/Cas9-based gene editing were awarded the Nobel prize in Chemistry.


Discovery helps engineer more accurate Cas9s for CRISPR editing

Scientists at the University of California, Berkeley and Massachusetts General Hospital have identified a key region within the Cas9 protein that governs how accurately CRISPR-Cas9 homes in on a target DNA sequence, and have tweaked it to produce a hyper-accurate gene editor with the lowest level of off-target cutting to date.

The protein domain the researchers identified as a master controller of DNA cutting is an obvious target for re-engineering to improve accuracy even further, the researchers say. This approach should help scientists customize variants of Cas9 – the protein that binds and cuts DNA – to minimize the chance that CRISPR-Cas9 will edit DNA at the wrong place, a key consideration when doing gene therapy in humans.

The Cas9 protein (gray) is an RNA-guided nuclease that can be programmed to bind and cut any matching DNA sequence (dark blue double helix), making it a powerful tool for genome engineering. Upon target binding, Cas9 protein domains undergo conformational rearrangements (the motions of individual amino acids are represented by rocket tails) to activate the Cas9-sgRNA complex for target cleavage. The REC3 domain (teal) is responsible for target sensing, which signals the outward rotation of the REC2 domain (magenta) to open a path for the HNH nuclease domain (yellow). This active conformation of Cas9 is then capable of triggering concerted cleavage of both strands of the target DNA. (Janet Iwasa graphic)

One strategy to achieve improved accuracy is to create mutations in the governing protein domain, called REC3, and see which ones improve accuracy without impacting the efficiency of on-target cutting.

“We have found that even minor alterations in the REC3 domain of Cas9 affect the differential between on- and off-target editing, which suggests that this domain is an obvious candidate for in-depth mutagenesis to improve targeting specificity. As an extension of this work, one could perform a more unbiased mutagenesis within REC3 than the targeted mutations we have made,” said co-first author Janice Chen, a graduate student in the lab of Jennifer Doudna, who co-invented the CRISPR-Cas9 gene-editing tool.

Co-first authors Chen, Yavuz Dagdas and Benjamin Kleinstiver, and their colleagues at UC Berkeley, Massachusetts General Hospital and Harvard University report their results online today in advance of publication in the journal Nature.

Hyper-accurate Cas9

Since 2012, when Doudna, a professor of molecular and cell biology and of chemistry and a Howard Hughes Medical Institute investigator at UC Berkeley, and colleague Emmanuelle Charpentier at the Max Planck Institute for Infection Biology repurposed the Cas9 protein to create a cheap, precise and easy-to-use gene editor, researchers have sought to lessen the chances of off-target editing. While improved fidelity benefits basic research, it is absolutely critical when editing genes for clinical applications, since any off-target DNA cutting could disable key genes and lead to permanent, unexpected side effects.

Within the last two years, two teams engineered highly accurate Cas9 proteins – an enhanced specificity one called eSpCas9(1.1) and a high-fidelity one called SpCas9-HF1 – and Chen and Doudna sought to learn why they cut with higher specificity than the wild-type Cas9 protein from Streptococcus pyogenes used widely today.

Currently, researchers using CRISPR-Cas9 create a single-guide RNA (sgRNA) – an RNA molecule that includes a chain of 20 ribonucleic acids that complements a specific 20-nucleic-acid DNA sequence they want to target — and attach it to Cas9. This guide RNA allows Cas9 to home in on the complementary DNA, bind to it and cut the double stranded helix. But the Cas9-sgRNA complex can also bind to DNA that doesn’t exactly match, leading to undesirable off-target cutting.

In 2015, Doudna’s lab discovered a conformational switch of Cas9 that is activated when the RNA guide and DNA target match. They found that only when the RNA and DNA match closely does the 3D structure of Cas9, in particular the conformation of the HNH nuclease domain, change and activate the scissors of Cas9. However, the process responsible for sensing the nucleic acids upstream of the conformational switch remained unknown.

In the current study, Chen and Dagdas used a technique called single-molecule FRET (Förster resonance energy transfer) to precisely measure how the various protein domains in the Cas9-sgRNA protein complex – in particular REC3, REC2 and HNH – move when the complex binds to DNA.

Maintaining efficiency while upping fidelity

They first determined that the specificity benefits conferred by eSpCas9(1.1) and SpCas9-HF1 could be explained by the fact that the threshold for the HNH conformational switch was much higher for these Cas9 variants than for the wild-type Cas9 protein, making the eSpCas9(1.1) and SpCas9-HF1 variants less likely to activate the scissors when bound to an off-target sequence.

Next, they uncovered that the REC3 domain is responsible for sensing the accuracy of target binding, which then signals the outward rotation of the REC2 domain to open a path for the HNH nuclease domain, activating the scissors. This active conformation of Cas9 is then able to cleave both strands of the target DNA.

Chen, Dagdas and Kleinstiver then showed that by mutating parts of REC3, it is possible to change the specificity of the Cas9 protein so that the HNH nuclease is not activated unless the guide RNA and target DNA match is very close. They were able to engineer an improved hyper-accurate Cas9, dubbed HypaCas9, that retains its on-target efficiency but is slightly better at discriminating between on- and off-target sites in human cells.

“If you mutate certain amino acid residues in REC3, you can tweak the balance between Cas9 on-target activity and improved specificity we were able to find the sweet spot where there is sufficient activity at the intended target but also a large reduction in off-target events,” Chen said.

By continuing to explore the relationships between structure, function and dynamics of Cas9, Doudna and her team hope to further engineer the protein with exquisite sensitivity to reliably and efficiently perform a variety of genetic alterations.

Co-authors of the paper include Keith Joung, a professor at Harvard and Mass General, whose lab engineered the high-fidelity Cas9, SpCas9-HF1 Ahmet Yildiz, a UC Berkeley associate professor of molecular and cell biology and of physics graduate student Lucas Harrington and former postdoc Samuel Sternberg of UC Berkeley and Moira Welch and Alexander Sousa of Mass General.

The work was supported by the National Institutes of Health (GM094522, GM118773, R35 GM118158) and the National Science Foundation (MCB-1617028).


By Sharon Tregaskis

This article originally appeared in the 2019 VP&S Annual Report.

The current regulatory environment varies among nations, with the legality of human gene modification depending heavily on a clinician’s geography, raising the possibility that scientists with ambitions curtailed in their home countries might move to more favorably regulated environs. And, no consensus has emerged on the mechanisms that might be used to impose international standards for how CRISPR is used. “There are a variety of possibilities,” says Appelbaum. “You could have legislation that controls or proscribes use of CRISPR or other gene-editing technologies, you could have voluntary self-regulation by the research community, or rules imposed by funders, or a completely unregulated environment, in which researchers and clinicians are free to do what they want with technology that’s available to them.”

As the technology advances, Appelbaum anticipates that society will be forced to confront profound questions about what it means to be human. “The assumption that we can identify conditions that should be extirpated from the human gene pool—assuming that were possible, which given the heterogeneous bases for many conditions is extremely unlikely—makes the question of whether it would be desirable a real one.” Consider, he suggests, the enormous creativity in mathematics demonstrated by some individuals on the autism spectrum or the cultural contributions of artists afflicted by mood disorders. “There are questions of neurodiversity,” he says, “but also the reality that the same gene, the same variant may have multiple consequences, particularly when we’re talking about complex traits.” And, he notes, it may be impossible to fully comprehend the choices we confront. “As we begin to be able to edit the gene pool it may be the case that we can’t anticipate some of the consequences of the changes we’re making.”


1. Introduction

Vaccination has saved hundreds of millions of lives, and has had spectacular success in eliminating smallpox and in greatly reducing the burden of infections such as yellow fever, diphtheria, meningitis and measles. Despite this impressive record, the development of vaccines against global pandemics such as HIV, TB, malaria and dengue is faced with major challenges. Among the major scientific challenges are the difficulties in identifying the relevant antigens that can be incorporated into a vaccine. A second major challenge is in defining the quality of the immune response that confers protection against infection, and in determining the mechanisms by which the immune system mounts the protective response. The latter is crucial for evaluating which immunological parameters are stimulated by vaccination (i.e. correlates of immunogenicity), or are associated with protection against subsequent infection, as determined in clinical trials (i.e. correlates of protection). The final challenge is to devise strategies to induce protective immunity that is long lasting. Recent developments in the field of systems biology offer the tools to analyse the dynamics and interactions of all components of a biological system during vaccination. These systems-level analyses are beginning to define the molecular correlates (‘signatures') of immunity and protection, and are yielding new insights into the mechanisms by which vaccines induce protective immunity [1𠄴]. Here, we review these advances and their promise in enabling the development of vaccines against unmet medical needs.


RESULTS

Design of HaitiV

We used nine different modifications to derive the new vaccine, HaitiV (Table 1), and whole-genome sequencing confirmed that all planned mutations were present. Mutations were engineered to ensure biosafety while maintaining HaitiV’s capacity for intestinal colonization so that, like wild-type (WT) V. cholerae and some previously tested live vaccine candidates (7, 11), it may impart long-term immunity after a single oral dose. To ensure the safety of HaitiV, we removed the bacteriophage (CTXΦ) encoding CT (Fig. 1A) (12), the pathogen’s principal virulence factor, and provided stringent impediments to toxigenic reversion. The boundaries of the CTXΦ deletion remove a sequence necessary for its chromosomal integration as well as the gene encoding a multifunctional toxin (MARTX) (13). In addition, HaitiV lacks hupB, a gene necessary for episomal maintenance of CTXΦ (14). Further vaccine engineering included steps to (i) reduce potential vaccine reactogenicity by deleting V. cholerae’s five flagellins (15) (ii) eliminate the vaccine’s capacity to transfer genes, conferring resistance to antibiotics, that lie within the SXT integrative conjugative element (ICE) (Fig. 1B) (iii) allow the vaccine to produce the nontoxic B subunit of CT (fig. S1), an antigen that may elicit protection against diarrheal disease caused by enterotoxigenic Escherichia coli (ETEC) as well as V. cholerae (16) and (iv) minimize potential gene acquisition by deleting recA, thereby markedly reducing the strain’s capacity for DNA recombination. HaitiV also encodes a clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) system specifically targeting the toxin gene ctxA. The CTXΦ bacteriophage bearing intact ctxA was unable to infect the vaccine harboring the CRISPR/Cas9 system, whereas a CTXΦ variant lacking ctxA showed no such barrier to infection (Fig. 1C).

Genetic alterations in HaitiV, a live attenuated cholera vaccine.

(A) Deletion of the CTX prophage and adjacent sequences, including the satellite prophages TLC and RS1 and MARTX toxin genes (yellow area, deleted region). (B) Deletion of genes conferring resistance to trimethoprim (dfrA), sulfamethoxazole (sul2), streptomycin (strAB), and chloramphenicol (floR). (C) An anti-ctxA CRISPR system provides immunity to CTXΦ infection: Streptococcus pyogenes cas9 along with sequence encoding a single-guide RNA (sgRNA) targeting ctxA, integrated into the HaitiV lacZ locus schematic showing targeting of the CTXΦ genome by the anti-ctxA Cas9-sgRNA complex HaitiV with/without the CRISPR system (CRISPR +/− ) were infected with either CTXΦ-IGKn (Target+ intergenic Kan R cassette, intact ctxA) or CTX-KnΦ (Target− ctxA replaced by Kan R cassette), and the number of transductants was monitored. No detectable Kan R transductants shown as “†”.

Intestinal colonization by HaitiV

Comparative studies of orogastrically inoculated HaitiV or the WT V. cholerae isolate from which it was derived (referred to as HaitiWT) were performed in infant rabbits, a small-animal model that recapitulates many aspects of human cholera, including rapid mortality (17). All animals inoculated with HaitiWT progressed to a moribund state by 18 hours post inoculation (HPI). Upon necropsy, the ceca of these animals were filled with fluid (Fig. 2A) previously found to resemble ctxAB-dependent choleric diarrhea (17). In marked contrast, minimal or no fluid had accumulated by 18 HPI in the ceca of littermates inoculated with HaitiV (Fig. 2A). Animals inoculated with HaitiV did not exhibit cholera-like illness during observation periods extending to 90 HPI, although in rare cases animals showed mild and self-limited noncholeric diarrhea. Animals inoculated with HaitiV continued to gain weight up to 90 HPI, providing further indication that HaitiV inoculation is not detrimental to overall health or development (Fig. 2B).

(A) Fluid accumulation ratios (FARs) after littermates were pretreated with ranitidine-hydrochloride, to reduce stomach acidity, and inoculated with either WT (n = 11) or HaitiV (vaccine n = 10). Plots show mean and SD derived from two litters. ****P < 0.0001, unpaired t test. (B) Successive daily weights of animals inoculated with 10 9 colony-forming units (CFU) of HaitiV (n = 10). (C) WT (blue circles) or HaitiV (red squares) CFU recovered from rabbit dSIs at day 1 or day 4 after inoculation (each of the three groups consists of animals from at least two litters). Lines indicate geometric means, and the open symbol indicates the limit of detection for the single animal from which no CFU were recovered. N.S. (not significant): P ≥ 0.05, Kruskall-Wallis test followed by Dunn’s multiple comparisons test. (D) Competitive indices (CIs) of dSI bacteria 1 day after inoculation with a 1:1 mixture of WT and HaitiV. The open symbol indicates limit of detection for the single animal from which no vaccine CFU were recovered lines and bars indicate geometric means and geometric SD of CIs across two litters (n = 6). (E) WT (blue) and HaitiV (red) CFU recovered from coinoculated animals. The open symbol indicates the limit of detection the single animal from which no vaccine CFU were recovered, and lines indicate geometric means.

The distinct responses to HaitiWT or HaitiV inoculation were not associated with differences between intestinal colonization by the two strains. At 18 HPI, there was no statistically significant difference in V. cholerae colonization of the distal small intestine (dSI) between littermates inoculated with HaitiV or HaitiWT (Fig. 2C). HaitiV burden showed no reduction by 90 HPI (Fig. 2C), indicating that prolonged intestinal colonization by HaitiV does not cause disease. Although intestinal colonization by HaitiV and HaitiWT was not statistically distinguishable in single inoculation experiments, when animals were coinoculated with a 1:1 mixture of HaitiWT and HaitiV, the WT strain outcompeted the vaccine strain (Fig. 2D).

HaitiV-mediated colonization resistance

HaitiV’s robust occupancy of the intestine motivated us to test whether HaitiV-colonized animals might exhibit resistance to colonization by HaitiWT even before the development of an adaptive immune response due to, for example, alteration of the pathogen’s intestinal niche. To test this possibility, we inoculated animals with either HaitiV (live vaccine), formalin-killed HaitiV (killed vaccine), or a buffer control (mock), then challenged them 24 hours later with a lethal dose of HaitiWT. Animals in the buffer and formalin groups developed severe cholera-like illness after HaitiWT challenge, and intestinal burdens of HaitiWT in these animals resembled those without pretreatment (Fig. 3A). Conversely, no animals that received live vaccine exhibited signs of severe disease within 18 hours of HaitiWT challenge, and lower burdens of HaitiWT were recovered from the intestines of animals previously inoculated with live vaccine versus those inoculated with killed vaccine (Fig. 3B). The reduction in HaitiWT burden varied in magnitude across animals inoculated with live vaccine, with the burden falling below the limit of detection in two animals. The live vaccine’s antagonism of HaitiWT colonization (that is, colonization resistance) appears dependent on previous inoculation of HaitiV normal burdens of HaitiWT were observed in animals inoculated with the two strains simultaneously rather than sequentially (compare Figs. 2E and 3B).

(A) WT CFU (blue circles) recovered from the dSI of animals 18 hours after inoculation with WT. Littermates were pretreated with sodium bicarbonate buffer (mock, n = 8) or formalin-killed HaitiV (killed vaccine, n = 7) 24 hours before WT challenge geometric means of each group across three litters are shown. N.S.: P ≥ 0.05, Mann-Whitney test. HaitiV and HaitiWT (B) or N16961 WT (C) CFU recovered from the dSI of animals 18 hours after challenge. Animals were pretreated with killed (n = 6) or live (n = 8) vaccine 24 hours before challenge. Open symbols indicate limit of detection for five animals in which no CFU were recovered, and lines indicate the geometric mean of each group across two litters. ***P < 0.001, *P < 0.05, Mann-Whitney test. (D) HaitiTn CFU (blue circles) and unique transposon mutants (black triangles) recovered from the dSI of individual animals (rabbits r1 to r6) 1 day after inoculation of the transposon mutant library without pretreatment. (E) HaitiTn CFU (blue circles), HaitiV CFU (red squares), and unique transposon mutants (black triangles) recovered from the dSI of individual animals (rabbits r1 to r7) 18 hours after inoculation of the transposon mutant library. Animals were pretreated with HaitiV 24 hours before challenge with the transposon mutant library. (F and G) Results of Con-ARTIST (39) analysis for single-inoculation (rabbit r4) and sequential-inoculation (rabbit r6) samples with the largest number of unique genotypes. The x axis indicates the change in relative abundance of insertion mutants per gene in vivo, and the y axis indicates the concordance of independent insertion mutants within each gene. Genes exhibiting a greater than twofold change [log2 (mean fold change) <−1 or >1] across multiple mutants (mean inverse P > 10 2 ) are considered depleted or enriched. Enriched mutants cqsS and hapR are indicated in blue. Mutations in known colonization factors, including toxin-coregulated pilus biogenesis (red circles) and the associated transcriptional regulators toxR and toxS (red asterisks), were depleted.

To assess the specificity of colonization resistance, we repeated the vaccine study, challenging with V. cholerae N16961, an early El Tor strain administered to human volunteers in studies of cholera vaccine efficacy (7). The Haitian and N16961 strains were isolated independently and are of distinct serotypes (10). Animals inoculated with live HaitiV, but not killed HaitiV, also exhibited colonization resistance against the N16961 WT challenge (Fig. 3C), demonstrating that HaitiV-mediated colonization resistance is neither strain- nor serotype-specific.

The colonization resistance evident in animals inoculated with live HaitiV led us to hypothesize that the vaccine’s occupancy of the intestine interfered with processes required for colonization by the challenge strain. We performed a forward genetic screen to identify mutations that allow HaitiWT to resist or evade vaccine-mediated antagonism. Such mutations could provide insight into the mechanism(s) by which HaitiV mediates colonization resistance and were predicted to confer a fitness advantage to HaitiWT specifically in the HaitiV-colonized intestine thus, animals were challenged with a pooled HaitiWT transposon-insertion library (HaitiTn) in the absence of pretreatment (single inoculation Fig. 3, D and F) or 24 HPI with live vaccine (sequential inoculation Fig. 3, E and G). HaitiTn colonization in the absence of pretreatment was indistinguishable from HaitiWT colonization of animals previously inoculated with a mock treatment or killed vaccine (Fig. 3). In addition, the range of HaitiTn colonization in vaccine-pretreated animals recapitulated the highly variable HaitiWT burden observed upon sequential inoculation of HaitiV and HaitiWT (Fig. 3).

To identify enriched mutants, we sequenced the transposon junctions from HaitiTn recovered from the dSI and performed a genome-wide comparison of mutant abundance in animals subjected to HaitiTn challenge without pretreatment (Fig. 3F and table S3) or after HaitiV inoculation (Fig. 3G and table S4). To ensure requisite statistical power, we restricted our analysis to animals colonized by sufficiently diverse HaitiTn populations encompassing multiple independent disruptions per gene (rabbits r3 to r6 for single inoculation and rabbits r4 to r7 for sequential inoculation). Notably, insertions disrupting cqsS and hapR, components of a Vibrio-specific quorum sensing (QS) pathway, were enriched in multiple animals, independent of pretreatment (Fig. 3, F and G, fig. S2, and tables S3 and S4). QS down-regulates expression of virulence and colonization factors at high cell densities (1821), and enrichment of cqsS and hapR mutants, which are blind to this inhibition, suggests that QS pathways constrain HaitiWT growth in the intestine. Corresponding enrichment of QS mutants was not identified in similar analyses of closely related V. cholerae isolates (22), suggesting that QS may play a distinct role in the pathogenesis of variant El Tor strains. Our genome-wide screen failed to identify any mutants that were consistently and specifically enriched in vaccine-colonized animals, indicating that single loss-of-function mutations are unlikely to enable HaitiWT to resist or evade vaccine-mediated antagonism.

The genetic diversity intrinsic to the HaitiTn library used above allowed us to assess whether HaitiV-mediated colonization resistance was associated with changes in the severity of the infection bottleneck that V. cholerae encounters in vivo (23, 24). V. cholerae recovered from the intestine arise from a founding population of organisms that persist after a stochastic constriction of the bacterial inoculum (23), and the severity of this infection bottleneck can be estimated from the number of unique transposon-insertion mutants recovered from the intestine (25). A subset of animals previously inoculated with live vaccine were colonized by relatively few unique insertion mutants and showed low HaitiTn burdens (Fig. 3E, rabbits r1 to r3), suggesting that HaitiV-mediated colonization resistance is, in some cases, associated with a highly restrictive infection bottleneck. There was no overlap in the mutants recovered from low-diversity animals, which indicates that the restrictive infection bottlenecks observed in some HaitiV-inoculated animals are stochastic and genotype-independent. We also observed reduced colonization in animals in which the vaccine did not appear to impose a bottleneck (Fig. 3D, rabbits r4 to r7). The variable bottlenecks observed in vaccine-colonized animals, along with our inability to identify mutants resistant to vaccine-mediated antagonism, highlights the possibility that the mechanism(s) underlying colonization resistance may be complex and/or multifactorial. However, the lower burdens of HaitiWT and the absence of severe disease after challenge in vaccine-colonized animals suggest that inoculation with HaitiV may be sufficient to protect against cholera-like illness.

HaitiV-induced rapid protection against cholera-like illness

To quantify HaitiV-dependent protection from cholera-like disease, we inoculated infant rabbits with live or killed vaccine, challenged them 24 hours later with HaitiWT, and conducted blinded hourly monitoring to assess their status. All animals inoculated with killed vaccine developed diarrhea (median onset, 15 hours) and progressed to a moribund state within 29 hours of HaitiWT inoculation (median, 18.8 hours) (Fig. 4A). In contrast, animals inoculated with live vaccine were significantly slower (P < 0.01, log-rank test) to develop diarrhea (median, 28.3 hours one animal did not develop diarrhea) and showed a marked increase in survival time after lethal challenge (median, >41.3 hours Fig. 4A) and in survival time after onset of diarrhea (>13 hours versus 5 hours in control animals Fig. 4B). In addition, four animals inoculated with live vaccine had not reached a moribund state when the study was concluded 41 hours after lethal challenge despite detectable HaitiWT colonization in all animals (Fig. 4, A and C). Thus, HaitiV may protect from disease even in the absence of absolute colonization resistance. The rapidity of HaitiV-induced colonization resistance and disease protection, and the observation of these phenotypes in a neonatal model of infection, are not consistent with vaccine-elicited adaptive immune protection. Instead, our data indicate that HaitiV colonizes the intestine and mediates viability-dependent protection against cholera, properties consistent with the definition of a probiotic agent (26).

(A) Survival curves tracking progression to moribund disease status in animals inoculated with WT at 0 hours after pretreatment (at t = −24 hours) with killed (black, n = 8) or live vaccine (red, n = 7). ***P < 0.001, log-rank test. (B) Disease progression from the onset of diarrhea to moribund status in animals, pretreated with killed (black, n = 7) or live vaccine (red, n = 6) that developed visible diarrhea. ***P < 0.001 and **P < 0.01, log-rank test. (C) WT CFU (blue circles) recovered from the dSI of animals 41 hours after challenge [from (A)] that did not progress to moribund disease status. (D) Effect of reactive vaccination on the number of cholera infections in a simulated outbreak (R0 = 2.1) starting with a single infection in a population of 100,000 susceptible individuals where the reactive vaccination campaign (RVC) is triggered once the number of symptomatic individuals reaches 1000 (1% of the total population), indicated by the dashed line. Rollout of doses is modeled with a constant rate over 7 days until 70% of the population is vaccinated, as achieved by recent RVCs. Modeling parameters are described in fig. S3B.

To investigate how rapid protection, like that elicited by HaitiV, might affect reactive vaccination campaigns, we modified a previously published mathematical model of a cholera outbreak in a susceptible population (27), an epidemic context prioritized for reactive OCV interventions (28). Our modifications to the model (fig. S3A) allowed us to compute the effects of hypothetical vaccines that confer equal degrees of protection in 1 day [fast vaccine, based on observations in Figs. 3 and 4 (A and B)] or in 10 days [slow vaccine, when some recipients of killed OCVs manifest vibriocidal antibody titers (29)]. The model does not account for transient protection and instead assumes that, once elicited, vaccine efficacy remains constant throughout the simulated epidemic. Varying different model parameters revealed that maximal benefit of a fast vaccine, relative to a slow vaccine, occurs under transmission dynamics consistent with recent outbreaks (R0 = 1.5 to 3) and with rapid vaccine administration (figs. S3B and S4). These simulations revealed that, compared to a slow vaccine, an equally efficacious fast vaccine could avert an additional 20,000 infections in a population of 100,000 (Fig. 4D) by preventing infections that could be acquired in the window between administration of the slow vaccine and the emergence of protective immunity.


CRISPR Scientist's Biography Explores Ethics Of Rewriting The Code Of Life

The Pfizer and Moderna COVID-19 vaccines are the first vaccines to be activated by mRNA — and would not have been possible without the invention of the gene editing technology known as CRISPR.

In his new book, The Code Breaker, author Walter Isaacson chronicles the development of CRISPR and profiles Jennifer Doudna, who, along with Emmanuelle Charpentier, won the 2020 Nobel prize in chemistry for their roles in developing the technology.

CRISPR has already led to experimental treatments for Huntington's disease and sickle cell anemia, as well as certain cancers. Isaacson likens its technological capabilities to "Prometheus snatching fire from the gods — or maybe Adam and Eve biting into the apple."

"The very secrets of life — our DNA — is something that we can not only read these days, but we can write. We can rewrite it if we want to," Isaacson says. "It made me think that all of us should understand and marvel at and be excited about this notion."

But, Isaacson warns that gene editing also raises a host of moral and ethical questions — especially as the technology becomes more developed.

"In the future, you might be able to do more complicated things — change hair color or muscle mass or memory cells in a human being," he says. "Like any technology, [it] is only as good or bad as we are. . So that's [why] we ought to have the discussion: What type of genes do we want to edit?"

Interview highlights

On how gene editing is next science revolution

I think in our modern times we've had three great revolutions: The first was the physics revolution. And it sort of starts at the beginning of the 20th century with Einstein's papers — and based on that fundamental kernel known as the atom. And from that, we get the atom bomb, and space travel and G.P.S. and semiconductors.

The second half of the 20th century was also based on a very small kernel of our existence, called the bit — meaning a binary digit. And it meant that all information could be coded in zeros and ones and binary digits. And that leads you to the internet and the microchip and the personal computer. And so that gives us the digital revolution, which dominates the second half of the 20th century.

Now we've come to another particle, a fundamental particle of our existence, which is the gene. And in the beginning of this century, in 2000 or so, we sequenced the entire human genome. And now with Jennifer Doudna and the things that she and her colleagues have invented, we found ways to rewrite that genome. And so this part of the 21st century, I think, will be a biotech revolution, a life sciences revolution.

On how the gene editing technology CRISPR mimics what the immune system does to bacteria

Whenever viruses attack certain bacteria, those bacteria do something very clever: They take a mugshot and they put it in their own genetic material of the bacteria. . So that when the virus attacks again, the bacteria remembers it and uses a little guide RNA and a pair of "scissors" (an enzyme that acts as a pair of scissors) and cuts up the invading virus. So that allows the bacteria to have an adaptive immune system, meaning every time a new wave of virus comes along, it takes that mug shot and will be able to fight it off the next time. And that's just what we, of course, need in this day when we're being attacked by wave after wave of viruses. .

What we can do and what Jennifer Doudna . discovered is that we can engineer this system that bacteria have and say, OK, we'll code in the place we want the DNA cut. So we can take this billion-year-old tool that bacteria have and reprogram it so we can aim at any sequence of DNA we want to change in our own bodies. If we want to change a gene, we can do so.

On CRISPR home testing kits, soon available to the public

There's going to be these wonderful CRISPR-based home testing kits that build directly on the discovery that [Jennifer Doudna and Emmanuelle Charpentier] won the Nobel Prize for, which is using this old technology that bacteria have, called CRISPR, to say, "I can spot any virus, just by reading the genetic code of things in my body." And so [eventually], we'll be able to have these [tests] in which, just in a minute, you can detect not only viruses or flu, you can detect bacteria, you could detect cancer, you can do your gut microbiome. That's going to be a great platform: these CRISPR home-based tests upon which people will build all sorts of wonderful medical tools. . These home testing kits will help bring biology into our home and bring us into a whole new era where we can do easy self-diagnosis of the things happening in our body.

On why the U.S. Defense Department funds research on gene editing

A lot of the research into CRISPR and genetic engineering has come from the Defense Department, usually as a defensive mechanism, like how would you make it so that radiation wouldn't be as harmful to your cells? That's just a genetic issue. Can you make cells that are more resistant to radiation? Can you make them more resistant to other forms of poisons or whatever?

The Defense Department also helped Jennifer Doudna and the people around her at Berkeley in the Bay Area create something called anti-CRISPR — because you could imagine a bad actor using this gene editing technology to do something harmful, to try to edit our genes, to do bad things. . Vladimir Putin was talking about CRISPR's gene editing technology to a youth group at one point and he said, "We might use it to make better and stronger soldiers that don't feel pain." So that you can realize, some of our enemies might be doing things. We got to figure out how to counteract it.

On gene editing getting into the wrong hands

I think that CRISPR and other gene editing technology are easy enough that it could be used by people who aren't playing by the same rules as a research scientist. . So I can imagine people saying, "I want to create tools that will help my memory [or] increase my muscle mass." But like any drugs or any tools, we hope that there'll be some types of regulation [and] that the Food and Drug Administration and others will say, "Here's what you can use it for and here's what you can't." .

When Jennifer Doudna first helped create [CRISPR] she had a nightmare and she walked into a room with somebody who said, "I want to understand your new technology." And the person looked up and it was Adolf Hitler. And this made her realize that if it got into the wrong hands, somebody might use it for eugenics purposes, might want to create a "master race," might want to create people who were stronger as soldiers. And she decided to gather scientists from around the world to say, let's figure out . the wonderful things this gene editing technology can do, but let's also try to limit things that would be inheritable or things that would not be as easy to control. And so she's been one of the leaders that said this is an important and good technology — let's make sure we don't misuse it.

Sam Briger and Seth Kelley produced and edited this interview for broadcast. Bridget Bentz, Molly Seavy-Nesper and Deborah Franklin adapted it for the Web.

This is FRESH AIR. I'm Terry Gross. The Pfizer and Moderna COVID vaccines are the first vaccines to be activated by mRNA. These vaccines build on the breakthroughs of the gene-editing technology known as CRISPR. This technology is also being used to treat people who have sickle cell anemia, certain cancers, Huntington's disease and congenital blindness, and will likely be used to treat many other diseases in the future. There are many other CRISPR-related breakthroughs on the horizon and a lot of moral and ethical questions to deal with about the editing of the basic element of human life.

One of the developers of CRISPR is Jennifer Doudna, who shared a Nobel Prize last year for her discoveries about gene editing. Doudna and the story of RNA-related scientific breakthroughs are the subjects of Walter Isaacson's new book, "The Code Breaker." While writing the book, he became part of a double-blind trial of the Pfizer vaccine. In other words, he was given the vaccine but wasn't told whether it was actually the vaccine or a placebo. Then he was monitored for symptoms of COVID and for side effects of the vaccine. Isaacson is also the author of biographies of Ben Franklin, Steve Jobs, Albert Einstein and Leonardo da Vinci. He's a professor of history at Tulane and was formerly the CEO of the Aspen Institute, chair of CNN and editor of Time magazine.

Walter Isaacson, welcome back to FRESH AIR. It is a pleasure to have you back on the show.

WALTER ISAACSON: Terry, it is so great to be back with you.

GROSS: So let's start with the vaccine. How did you have the option of participating in the Pfizer vaccine study? And why did you want to do it?

ISAACSON: I was fascinated about the science and the thrill that we might have a vaccine. And I really believe that we should all participate in science a little bit more, not be intimidated by it. So when I heard they were making vaccines and testing them that used RNA, I just went online at Ochsner Hospital down here in New Orleans and volunteered to be one of the participants in the clinical trial. And, boom, the next day, I got called up. And they gave me a shot, made me close my eyes to make sure I couldn't guess whether it was the real thing or the placebo. But it made me feel I was a participant in helping us figure out how to fight COVID.

GROSS: So when was this? This was last summer?

GROSS: OK (laughter) - memorable day. What was it like for you not knowing whether you got the real thing or a placebo?

ISAACSON: It was important not to know because I didn't want to change my behavior. You know, I still wore masks and did social distancing. But they monitored my blood. They made sure it was all going well. And they did tell me that at the end, if I'd gotten the placebo, they'd switch you over and monitor you with the real thing. So I was just waiting to see.

GROSS: How did they unblind you? In other word, how and when did they let you know what you really got? And what did you get?

ISAACSON: Well, it was six months later, after you do your final blood test. And then they unblind you. I had gotten the placebo. But at that point, they were rolling out the real Pfizer vaccine anyway. So I was able to get the real thing.

GROSS: Well, that's great. Did you have side effects to report?

ISAACSON: No. In fact, I was kind of worried. Even with my second shot, I kept thinking, everybody's got side effects. Maybe they gave me the placebo again. But nope.

ISAACSON: I didn't really have any side effects.

GROSS: Before we get deep into the science of the vaccine itself, let's do some background science and background history of RNA so it'll make it easier to understand the science of the vaccine. So we're talking here about RNA or, more specifically, mRNA. RNA is a sister of DNA. We know what DNA is kind of. I mean, we know that we can submit our DNA through saliva and find out more about our genealogy. We know if there was a crime, they could take DNA samples and trace who the criminal is through a DNA databank if you're lucky and the DNA is already in the databank. So what is RNA compared to DNA?

ISAACSON: You're right. DNA is the famous sibling. It's the one that gets on the magazine covers. And we talk about the DNA of an organization, of a society. But like a lot of famous siblings, DNA doesn't do a whole lot of work. It just sits there in the nucleus of our cell guarding our genetic information. The real work is done by RNA. The RNA goes in there, takes copies of a particular gene that might be needed and then goes to that region of the cell where you make proteins. And it's the RNA that oversees the making of the protein. And that work of taking the code from in our cell's nucleus from the DNA and going to make protein, that's called the messenger work of RNA. And that's why these little snippets are called messenger RNAs. And when everybody was trying to race to study the human genome and do the sequencing of DNA, there were some scientists who said, let's look at this more interesting molecule, which, by the way, turns out to be able to replicate itself. And so - lo and behold, it's the beginning of all life on this planet. So RNA turns out to be far more interesting than its brother, DNA.

GROSS: And a recurring theme in your book is that nature is beautiful and that RNA is beautiful. What's beautiful about it?

ISAACSON: RNA is beautiful because it's so simple, which is a four-letter code. It can build any protein that's in our body. It was the molecule that started replicating itself three, 4 billion years ago in this stew that was on our planet. And that's how life begins on our planet. And RNA can serve as a guide to help cut up pieces of DNA, which is what gene-editing technology is all about. Or it could serve as a messenger to say, hey build this protein in the cell because that mimics a spike protein on a coronavirus. And that way, the person will be immune to coronavirus. And so RNA can do all these things, act as a guide for scissors, act as a messenger to build proteins. And it really does the daily work every, you know, time we need proteins built for anything, whether it's our hormones or our hair or our eyes or the little things that - the neurons in our brain.

GROSS: So CRISPR - spelled C-R-I-S-P-R - is an acronym for a big scientific term that I don't think you even need to mention because no one will understand what it means (laughter). But anyway, so CRISPR is a gene-editing tool. Before we get to how that uses RNA, what does it mean to be a gene-editing tool? What are some of the ways that's being used now?

ISAACSON: If you want to change the genes in our body, you can do it just by snipping them out and sometimes putting in a replacement. So let's say somebody has sickle cell anemia or Huntington's. That's a simple single-gene mutation. And so you can change it with a gene-editing tool. In the future, you might be able to do more complicated things, change hair color, a muscle mass or memory cells in a human being. And so what we do with gene-editing tools is we can fix diseases. And a little bit more controversially, we can edit the embryos of our children and make permanent changes in the human race.

GROSS: Yeah. And we'll talk about that a little later when we get to the moral and ethical questions surrounding this new genetic technology. So you describe CRISPR as an immune system that bacteria adapted when they get attacked by a new type of virus. So what's the relationship between the bacteria immune system and the gene-editing tool known as CRISPR?

ISAACSON: Whenever viruses attack certain bacteria, those bacteria do something very clever. They take a mug shot, and they put it in their own genetic material or the bacteria. And so in these bacteria, you see these clustered, repeated segments of DNA. And that's where the name CRISPR comes from.

So that when the virus attacks again, the bacteria remembers it and uses a little guide RNA and a pair of scissors - an enzyme that acts as a pair of scissors - and cuts up the invading virus. So that allows the bacteria to have an adaptive immune system, meaning every time a new wave of virus comes along, takes that mug shot and will be able to fight it off the next time. And that's just what we, of course, need in this day when we're being attacked by wave after wave of viruses.

GROSS: So how does that apply to gene editing?

ISAACSON: What we can do and what Jennifer Doudna, the hero of my book, discovered is that we can engineer this system that bacteria have and say, OK, we'll code in the place we want the DNA cut. And so we can take this old, billion-year-old tool that bacteria have and reprogram it so we can aim at any sequence of DNA we want to change in our own bodies. If we want to change a gene - clip - we can do so.

GROSS: You actually tried your hand at genetic editing (laughter) just to see, like, how it's done, how hard is it. Would you describe your experience as a non-scientist trying to do a genetic edit?

ISAACSON: I was in Jennifer Doudna's lab at Berkeley, and I figured out, all right, if I'm going to really write about this, I should learn to do by doing it. And so a couple of graduate students spent a couple of days with me, and we had test tubes and pipettes and those little centrifuges that spin things around. And we were able to take CRISPR and edit a human cell, put in a little phosphorescent gene in it so we could see it glow. And it wasn't really all that hard, which was a little bit exciting to me, but also a bit unnerving.

Now, don't worry, Terry. When we finished, we mixed it with chlorine and poured it down the drain so it didn't escape. But that's why we all have to be thinking about what are we going to do with these gene-editing tools because they're not all that complicated.

GROSS: Let's take a break. If you're just joining us, my guest is Walter Isaacson. His new book is called "The Code Breaker." We'll be right back after a break. This is FRESH AIR.

(SOUNDBITE OF TODD SICKAFOOSE'S "TINY RESISTORS")

GROSS: This is FRESH AIR. Let's get back to my interview with Walter Isaacson. His new book is called "The Code Breaker: Jennifer Doudna, Gene Editing And The Future Of The Human Race." So let's get to the new mRNA vaccines, the COVID vaccines. And we're talking about the Moderna and the Pfizer vaccines here. How is mRNA used in these vaccines?

ISAACSON: The most basic thing that RNA does on this planet is it serves as a messenger to take a piece of genetic code from the DNA that's in the nucleus of our cell and say build this protein. And the way it works in the vaccine is we know that the coronavirus has certain proteins that, if we can disrupt them, they ain't going to work anymore, such as the spike protein that's on the surface of the coronavirus. And so we can now just program in the genetic code that tells a snippet of RNA to go make a part of that spike protein so it becomes a facsimile of it, like a mug shot of it.

And then our immune system says, OK, I'm going to kick into gear whenever I see this thing, and I'll knock it out. And so that's what the vaccine does, is it stimulates our immune system to make sure that if that spike protein - the real thing - ever comes along, it's already got the antibodies to knock it out.

GROSS: So for people who are afraid that if they get vaccinated, they're going to get COVID - which they're not - what's the difference between what you're being inoculated with - the mRNA that you're being inoculated with and the actual virus, DNA or RNA?

ISAACSON: It used to be when we did vaccines, we would take the whole virus that we wanted to knock out, and we'd give the patient an inactivated version of that virus, so one that was a weakened version of the virus, whether it be polio or measles or mumps or rubella. And, you know, people feared, well, I'm getting a little of the virus in me. Is that going to be bad? It never was bad, but this is much safer.

You're not getting the real virus. You're just getting a tiny blueprint that tells your cell how to make a small bit of a protein that exists on the surface of the real virus. Now, that little protein that gets built - that's not going to hurt you. That's not the virus. That's just a tiny component of the virus. But it tells your immune system, if you ever see this little thing, this tiny component, knock it out. And that's the way your body learns to knock out the real virus.

GROSS: Now, some people do get some side effects, like they feel, like, headachy or a little flu-ish (ph) the next day, especially after the second dose. Some people get a rash after the first dose. These are not - these go away. They're not - having COVID or dying of COVID, that's far worse than having a problem for a day or two. But why would you get a reaction if you're not really getting a virus?

ISAACSON: Your immune system is starting to kick in. Your immune system is saying, all right, I'm going to fight off this tiny little piece of protein that the vaccination is telling my body to make. And whether it's a flu vaccine or any vaccine, when your immune system jumps into action, it can cause a bit of a headache or a little bit of a swelling. But you don't have the real virus. You just have your immune system getting to work. In fact, it's a good thing. When my wife got the second shot, you know, she had a headache for a few hours, a little bit of swelling. And I didn't. And I thought, oh, wow, her immune system's a lot better than mine. It's kicking in fast. So you actually want to feel a little something because that says, hey your immune system's working.

GROSS: So since you're explaining the science behind the vaccine - with all these new variants coming our way, variants that - some of them are more contagious or, you know, more infectious or possibly more lethal. And the question is, how good, how effective will these new vaccines be against these new variants? So with the mRNA vaccines, the Pfizer and the Moderna, what's the science behind how well they would adapt to a new variant?

ISAACSON: They've adapted very well, these mRNA vaccines, to the new variants that have come along because all of these variants still have the spike protein. And that's like a big barn door that the RNA vaccines and some other vaccines say, hey watch out for this spike protein. And knock it out if it comes along. So - so far, we haven't seen variants that don't get affected by these vaccines. These vaccines are pretty effective. But here's the cool thing, Terry, these RNA vaccines are easily coded and recoded. In other words, it's just like rewriting a word document or recoding a program.

If we have a major new variant of the coronavirus that has a spike protein that's a different shape, it's real simple just to say, all right, let's take the genetic code of this new variant of the coronavirus, and let's program a messenger RNA that will build its spike protein. And that would take, maybe, two or three days to just do the recoding. And so it's not all that hard. And then it would take a few weeks to start manufacturing the new vaccine. So what we've seen this past year is a major shift in the balance of power between us and viruses because we can just recode. Every time the virus mutates and changes, we can pretty quickly recode these messenger RNA vaccines so that they'll go after each new variant. Fortunately, we don't need to do so much yet because the current vaccines are very good at the variants that we are now facing.

GROSS: I thought some scientists weren't so sure about, for instance, the variant in Brazil and how effective the vaccine was going to be against that.

ISAACSON: Well, we've seen that it's proven so far to be pretty darn effective. But as I say, all coronaviruses, they mutate a lot. There'll probably be thousands of variants that will change. And at some point, we might get one that will totally evade the vaccinations we have. And that just means we'll have to have booster shots that we do pretty simply just by redoing the code for the messenger RNA we're using.

GROSS: So it's interesting that recoding the vaccine isn't hard. You can change the formula, so to speak, in a couple of days by rewriting the code. But then the manufacturing and the distribution becomes the issue. How quickly do you think they can gear up for that for boosters?

ISAACSON: Well, as you say, molecules are the new microchips. We can recode them pretty simply. Then, of course, you do have to make the solutions and put it in the vials and distribute it. But now that we've gone through this process of doing it, we know it can be done in months rather than years. And so I think we'll be prepared just like we are every flu season when we create new versions of the flu vaccine. We'll be prepared to do booster shot and new variations of our vaccines as a coronavirus emerges, which is why I do think that this is a turning point in the hundreds of millions of years that organisms on this planet have been fighting off viruses.

GROSS: We've talked about the Moderna and Pfizer vaccines. Is the Johnson & Johnson vaccine also related to RNA?

ISAACSON: The Johnson & Johnson vaccine is more of a genetically engineered vaccine. It has a gene that will once again create a immunity towards the spike protein of the coronavirus. But it's not done using RNA. It's done using the entire gene of a particular coronavirus.

GROSS: Let's take a short break here, and then we'll talk some more. If you're just joining us, my guest is Walter Isaacson. His new book is called "The Code Breaker: Jennifer Doudna, Gene Editing, And The Future Of The Human Race." We'll be right back. I'm Terry Gross, and this is FRESH AIR.

(SOUNDBITE OF KENNY BARRON AND DAVE HOLLAND'S "DR. DO RIGHT")

GROSS: This is FRESH AIR. I'm Terry Gross. Let's get back to my interview with Walter Isaacson. He's written biographies of Einstein, Da Vinci, Steve Jobs, Ben Franklin and Henry Kissinger. His new book "The Code Breaker" tells the story of scientific and medical breakthroughs through new understanding of RNA. The book is part science history and part biography of Jennifer Doudna, who won a Nobel Prize last year for her RNA-related discoveries about gene editing, which led to the gene-editing tool known as CRISPR. Her breakthroughs in our understanding of RNA also paved the way for the mRNA COVID vaccines created by Moderna and Pfizer.

When Jennifer Doudna, who as you say is the hero of your book, started researching RNA, it wasn't to make a vaccine or to create a gene-editing tool it was just to see what RNA could do, like what is RNA, because it was DNA that was getting all the attention. Tell us more about why she was so interested in RNA.

ISAACSON: That's one of the beautiful things about science, is that basic curiosity without any particular point - it's not like you're trying to invent something - but you're just basically curious about something in nature. It often leads to an amazing thing. And so Jennifer Doudna was just curious about how does RNA work? What is its shape like? How did it help begin life on this planet? And so she became interested, too, in how you could use RNA as a tool, as a guide, to help edit genes. And when she saw that's how the CRISPR system worked, it suddenly dawned on her - wow, this is not just basic curiosity this could be a very useful tool in the medical toolbox we have. It's what we call moving something from bench to bedside - in other words, a bench in the lab to the bedside of the patient.

GROSS: And she had been an academic. She was at the University of California, Berkeley, and had a lab there. And at some point, she wanted to kind of get her research out of the lab, like you're saying, and make it a practical tool in the real world. What was her first step in that direction?

ISAACSON: After she discovers how CRISPR can be used as a tool to edit genes, she forms a couple of companies. One is called Mammoth, and it becomes a detection technology for viruses. Well, you know, that's pretty useful these days. She formed it five or six years ago. But soon they'll be producing home kits that will test us for any virus. She also helped form a company called Caribou which uses this gene-editing tool as a therapy to help fix diseases and genetic diseases. So one of the things I like so much about Jennifer Doudna is she's interested in the basic science. She's excited about the beauty. She's curious about how nature worked. But she also likes to take the next step and say, after we've discovered something, how can we make it useful? How can we apply it to our own lives?

GROSS: Are the home testing COVID kits that are going to be available soon - or maybe already are available I've kind of lost track - are they building on her science?

ISAACSON: There's going to be these wonderful CRISPR-based home testing kits that build directly on the discovery that she won the Nobel Prize for, which is using this old technology that bacteria have called CRISPR to say, I can spot any virus just by reading the genetic code of things in my body, in which just in minutes, you can detect not only viruses of flu, you can detect bacteria, you could detect cancer, you can do your gut microbiome. That's going to be a great platform, these CRISPR home-based tests, upon which people will build all sorts of wonderful medical tools.

It'll be like your iPhone helps you bring apps into your home - these type of CRISPR-based home testing kits, being done by people at MIT and Harvard, but also by people who work with Jennifer Doudna out at Berkeley - these home testing kids will help bring biology into our home and bring us into a whole new era, where we can do easy self-diagnosis of the things happening in our body.

GROSS: Well, let's back up a second. You mentioned cancer in this list. We're going to be able to diagnose if we have certain cancers?

ISAACSON: Yeah, cancer tumors have genetic code, just as everything does, in terms of living things, just like coronaviruses do or bacteria do. So once you're able to develop a technology that can just go in and check to see if a particular piece of genetic code is around, then you'll be able to sequence tumors. You'll be able to sequence coronaviruses. And you'll say, hey I want a tool that will detect it. It won't be a home kit to detect tumors by the end of this year, but in the lab, that's already being done.

GROSS: Yeah, you describe, like, gene editing and the discovery of how mRNA works as being the equivalent of a new revolution in science. Give us your short summaries of the three revolutions with gene editing, with, you know, this kind of biology being the third.

ISAACSON: I think in our modern times, we've had three great revolutions. The first was the physics revolution, and it sorts of starts at the beginning of the 20th century with Einstein's papers, and it's based on that fundamental kernel known as the atom. And from that, we get the atom bomb and space travel and GPS and, you know, semiconductors. Second half of the 20th century was also based on a very small kernel of our existence called the bit, meaning a binary digit, and it meant that all information could be coded in zeros and ones and binary digits. And that leads you to the Internet and the microchip and the personal computer. And so that gives us the digital revolution, which dominates the second half of the 20th century.

Now we've come to another particle, a fundamental particle of our existence, which is the gene. And in the beginning of this century, in 2000 or so, we sequenced the entire human genome. And now with Jennifer Doudna and the things that she and her colleagues have invented, we found ways to rewrite that genome. And so this part of the 21st century, I think, will be a biotech revolution, a life sciences revolution, in which we'll be able to rewrite the code of life.

GROSS: Let's take a short break here, and then we'll talk some more. If you're just joining us, my guest is Walter Isaacson, and his new book is called "The Code Breaker." We'll be right back after a break. This is FRESH AIR.

(SOUNDBITE OF ALLEN TOUSSAINT'S "EGYPTIAN FANTASY")

GROSS: This is FRESH AIR. Let's get back to my interview with Walter Isaacson. His new book is called "The Code Breaker: Jennifer Doudna, Gene Editing, And The Future Of The Human Race."

So early on for Jennifer Doudna, when she was working on RNA and gene editing, she got a contract from the Defense Department to study if gene editing could be used to treat radiation sickness that would be caused by, you know, a nuclear device, an atomic weapon of one sort or another. That seemed really remarkable to me. I mean, I grew up in the shadow of, like, nuclear terror (laughter), you know, post-World War II, like, nuclear terror.

And the thought - and there are so many movies that have come out where people are suffering from radiation sickness, you know, like, what-if-there-was-a-nuclear-bomb kind of movies, where you watch people for the whole movie, like, suffer nuclear radiation sickness. And the thought that it's possible that gene editing could be used to cure that just seemed absolutely remarkable to me. So I don't know how far she got with that. Like, where do we stand with that aspect of her research?

ISAACSON: A lot of the research into CRISPR and genetic engineering has come from the Defense Department, usually as a defensive mechanism. Like, how would you make it so that radiation wouldn't be as harmful to your cells? That's just a genetic issue that - you know, can you make cells that are more resistant to radiation? Can you make them more resistant to other forms of poisons or whatever?

And the Defense Department also helped Jennifer Doudna and the people around her at Berkeley and the Bay Area create something called anti-CRISPR because you could imagine a bad actor using this gene-editing technology to do something harmful, to try to edit our genes to do bad things. And so you want to say, how do we reverse the process? So a lot of the money has come from the National Science Foundation. A lot comes from these foundations that support research into bad diseases, like Huntington's and sickle cell anemia. And then some of it comes from the Defense Department that wants to help defend against people who might use gene editing to attack us or to help make people less resistant to other forms of attack.

I mean, you know, Vladimir Putin was talking about CRISPR as a gene-editing technology to a youth group at one point, and he said, well, we might use it to make better and stronger soldiers that don't feel pain. So that's - you can realize, all right, some of our enemies might be doing things we got to figure out how to counteract it.

GROSS: So let's talk more about nefarious and unethical ways that this new technology can be used. One of the concerns you bring up in the book is biohacking. Describe what that - what those concerns are.

ISAACSON: Well, I think that CRISPR and other gene-editing technology are easy enough that they could be used by, you know, people who aren't playing by the same rules as a research scientist. It could be done in garages. So I can imagine people saying, hey I want to create tools that will help my memory, increase my muscle mass. But like any drugs or any tools, we hope that there'll be some types of regulation, that the Food and Drug Administration and others will say, here's what you can use it for and here's what you can't.

GROSS: Are there other, like, nefarious uses that scientists are worried about?

ISAACSON: When Jennifer Doudna first helped create this tool called CRISPR that allowed you to do gene editing, she had a nightmare. And she walked into a room with somebody who said, I want to understand your new technology, and the person looked up and it was Adolf Hitler. And this made her realize that if it got into the wrong hands, somebody might use it for eugenics purposes, might want to create a master race, might want to create people who were stronger as soldiers.

And she decided to gather scientists from around the world to say, let's figure out what the promise and the wonderful things this gene-editing technology can do, but let's also try to limit things that would be inheritable or things that would, you know, not be as easy to control. And so she's been one of the leaders that said, this is an important and good technology let's make sure we don't misuse it.

GROSS: So describe what inheritable means in this context and why scientists now are kind of drawing the line at that kind of work.

ISAACSON: It's easy to use gene-editing technology to fix problems in a living patient. For example, just this past year, a woman in Mississippi who has sickle cell anemia had her genes edited so that she no longer suffers from sickle cell anemia. Nobody would be against that - that a patient knew what she was doing, gave informed consent. But also, a couple of years ago, a doctor in China in an unauthorized experiment edited early-stage embryos of what turned out to be two twin girls, and he used those edits to make it so that the twins, the designer babies they were, didn't have a receptor that allowed them to get the virus that causes AIDS.

Now, you can see why some people say, well, that's great we could help wipe AIDS, you know, from the human species. But the problem with that type of use of CRISPR is when you do it in reproductive cells, like early-stage embryos or eggs or sperm. It affects every cell in the body, and thus it's inherited by the children and all the descendants of the edits you've made. And that's a line you don't want to cross lightly, is making the type of edit that will not only affect a patient but also affect the entire human species and all their descendants.

One of the people in my book - a wonderful young kid named, you know, David Sanchez - has sickle cell anemia. And he's being treated for it, and they tell him about CRISPR and how it could make it so his children won't have sickle cell anemia. And David says, wow, that's really cool. But then he says, but I think it should be up to the kid to decide. And they said, well, what do you mean? Wouldn't you want to make sure your kids didn't have sickle cell anemia and that all their kids didn't? And he said, well, I think it should be up to each person to decide because even though sickle cell anemia has been really brutal to me, it taught me a lot of things. It taught me character. It taught me persistence. It taught me empathy and patience.

And so he, even as a 17-year-old, is able to make this distinction between using these wonderful technologies to cure patients who give their consent and want to have their genes edited to cure the disease and then crossing the line to make inheritable edits. Well, that's a line, as I say, that we don't need to do now and maybe we should be careful about ever doing.

GROSS: So I want to get back to the Chinese scientist who edited the genes and embryos of two twins so that they wouldn't be able to get HIV, they would be immune to that. He thought he would be a hero. He ended up with a big fine and a three-year prison sentence. What went wrong with his experiment that teaches us a lesson about what could go wrong with these kinds of things?

ISAACSON: Well, first of all, what the Chinese scientist did was unsafe. We're not ready yet to make these type of edits. And in fact, the children he produced are called mosaics, meaning some of their cells were edited and some weren't. So that's obviously a bad thing. The second thing is we don't know the unintended consequences. You get rid of that receptor that allows you to get the virus that causes AIDS, but maybe it means you're more susceptible to malaria, or West Nile virus. So we want to know what the intended and unintended consequences are.

But the real question is, once we figure out how to make it safe and that it's going to be reliable, then we still may want to pause and say, do we really want to make designer babies, to edit our kids? In my own personal opinion - and in the book, we go through it step by step because I think we all have to think through the cases and how it would work. Jennifer Doudna, the hero of my book, other people, myself, we each discuss each of the cases you'd do it.

I think you'd probably would want to make inheritable edits when it comes to things like Huntington's disease or sickle cell anemia or Tay-Sachs, especially if there's no alternative that's an easy medical fix for those things. But if you're just doing it to enhance children - say you want to make them taller or you want to change their hair color - that's where I think we have to draw the line for the time being at least.

GROSS: You live in New Orleans and teach at Tulane University. Last year in New Orleans, as I recall, there was a big spike in the coronavirus after Mardi Gras. What was that period like for you?

ISAACSON: I remember sitting on my balcony on Mardi Gras a year ago, and my balcony is in the French Quarter overlooking Royal Street. And it was a wild time. There were people - nobody had died at that point from coronavirus in the United States, but we knew about the pandemic. And some people were dressed up with Corona beer bottle costumes and viral heads and masks, making fun of the coronavirus. And boom, about three weeks later, it really hits the United States. There's a spike throughout the country, and we realize, OK, we're in a whole new era.

GROSS: Walter Isaacson, thank you so much for talking with us.

ISAACSON: You know, it's always great, Terry. I hope someday we'll be back in Philadelphia together.

GROSS: That would be great. I look forward to that. Walter Isaacson is the author of the new book "The Code Breaker: Jennifer Doudna, Gene Editing And The Future Of The Human Race." After we take a short break, Ken Tucker will review the new expanded version of Hailey Whitters' 2020 country album "The Dream," which he says is proof of how important she's become. This is FRESH AIR.

(SOUNDBITE OF ERROLL GARNER'S "IT'S ONLY A PAPER MOON") Transcript provided by NPR, Copyright NPR.


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