Gene-editing offers hope for people with hereditary disorder

A group of patients with a hereditary disorder have had their lives transformed by a single treatment of a breakthrough gene-editing therapy, according to the lead researcher.

The patients from New Zealand, the Netherlands and the UK have hereditary angioedema, a genetic disorder characterised by severe, painful and unpredictable swelling attacks.

These interfere with daily life and can affect airways and prove fatal.

Now researchers from the University of Auckland, Amsterdam University Medical Center and Cambridge University Hospitals have successfully treated more than ten patients with the CRISPR/Cas9 therapy, with interim results just published in a leading journal.

"It looks as if the single-dose treatment will provide a permanent cure for my hereditary angioedema patients' very disabling symptoms," says principal investigator Dr Hilary Longhurst, who is both a clinical immunologist at Auckland Hospital Te Toku Tumai and an honorary associate professor at the University of Auckland.

"Plus, of course, there is huge potential for development of similar CRISPR/Cas9 treatments for other genetic disorders."

Globally, it is estimated one in 50,000 people have hereditary angioedema, however, because it is rare, it is often not correctly diagnosed.

In the phase one study, there were no serious or lasting side-effects from the single infusion, which took place over two to four hours under clinical supervision from late 2021 and onwards.

The investigational therapy, called NTLA-2002, utilises in vivo CRISPR/Cas9 technology to target the KLKB1 gene, which is responsible for producing plasma prekallikrein.

By editing this gene, the therapy reduces the levels of total plasma kallikrein, effectively preventing angioedema (swelling) attacks.

The trial, published in the New England Journal of Medicine, demonstrated dose-dependent reduction in total plasma kallikrein protein with reductions of up to 95 percent achieved.

A mean reduction of 95 percent in angioedema attacks was observed across all patients through to the latest follow-up.

The patients from the initial study will be followed up for a further 15 years to continue to assess long-term safety and efficacy.

A larger and more robust, double-blinded, placebo-controlled phase two trial is under way and a Phase 3 trial is planned to start in the second half of 2024.

Dr Danny Cohn, from the Department of Vascular Medicine at the Amsterdam University Medical Center says these promising results are a step forward for this group of patients.

"We've never been closer to the ultimate treatment goal of normalising hereditary angioedema patients' lives and offering total control of the disease," says Dr Cohn.

Dr Padmalal Gurugama, consultant in clinical immunology and allergy at Cambridge University Hospitals, UK says the gene editing therapy has the potential to significantly improve patients' lives.

"Hereditary angioedema can cause patients severe swellings and intense pain which can be life-threatening as well as restricting normal activities, such as going to work or school.

"Because it is often misdiagnosed, many patients undergo unnecessary treatments and invasive procedures."

The therapy affects only the patient and is not passed onto their children, who still have an even chance of inheriting the disorder.

The studies have been funded by US company Intellia Therapeutics, which chose New Zealand to lead the research as, at that time -- late 2021, it had relatively fewer Covid-19 cases than other countries.

So far, the only approved CRISPR therapy, CASGEVY, is for sickle cell disease and beta thalassemia.

However, CASGEVY is an ex vivo CRISPR therapy, where the cells are taken from the patient and edited outside of the body and then reinfused, whereas NTLA-2002 is an in vivo CRISPR therapy, where the targeted gene editing occurs directly within the body.

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New AI tool makes speedy gene-editing possible

An artificial intelligence program may enable the first simple production of customizable proteins called zinc fingers to treat diseases by turning genes on and off.

The researchers at NYU Grossman School of Medicine and the University of Toronto who designed the tool say it promises to accelerate the development of gene therapies on a large scale.

Illnesses including cystic fibrosis, Tay-Sachs disease, and sickle cell anemia are caused by errors in the order of DNA letters that encode the operating instructions for every human cell. Scientists can in some cases correct these mistakes with gene editing methods that rearrange these letters.

Other conditions are caused, not by a mistake in the code itself, but by problems in how the cellular machinery reads DNA (epigenetics). A gene, which provides the recipe for a particular protein, often partners with molecules called transcription factors that tell the cell how much of that protein to make. When this process goes awry, over- or underactive genes contribute to diabetes, cancer, and neurological disorders. As a result, researchers have been exploring ways to restore normal epigenetic activity.

One such technique is zinc-finger editing, which can both change and control genes. Among the most abundant protein structures in the human body, zinc fingers can guide DNA repair by grabbing onto scissor-like enzymes and directing them to cut faulty segments out of the code.

Similarly, zinc fingers can also hook onto transcription factors and pull them toward a gene segment in need of regulation. By customizing these instructions, genetic engineers can tailor any gene's activity. A drawback, however, is that artificial zinc fingers are challenging to design for a specific task. Since these proteins attach to DNA in complex groups, researchers would need to be able to tell -- out of countless possible combinations -- how every zinc finger interacts with its neighbor for each desired genetic change.

The study authors' new technology, called ZFDesign, overcomes this obstacle by using artificial intelligence (AI) to model and design these interactions. The model is based on data generated by the screen of nearly 50 billion possible zinc finger-DNA interactions in the researchers' labs. A report on the tool is publishing online Jan. 26 in the journal Nature Biotechnology.

"Our program can identify the right grouping of zinc fingers for any modification, making this type of gene editing faster than ever before," says study lead author David Ichikawa, PhD, a former graduate student at NYU Langone Health.

Ichikawa notes that zinc-finger editing offers a potentially safer alternative to CRISPR, a key gene-editing technology with applications that range from finding new ways to kill cancer cells to designing more nourishing crops. Unlike the entirely human-derived zinc fingers, CRISPR, which stands for clustered regularly interspaced short palindromic repeat, relies on bacterial proteins to interact with genetic code. These "foreign" proteins could trigger patients' immune defense systems, which may attack them like any other infection and lead to dangerous inflammation.

The study authors add that besides posing a lower immune risk, the small size of zinc-finger tools may also provide more flexible gene therapy techniques compared with CRISPR by enabling more ways to deliver the tools to the right cells in patients.

"By speeding up zinc-finger design coupled with their smaller size, our system paves the way for using these proteins to control multiple genes at the same time," says study senior author Marcus Noyes, PhD. "In the future, this approach may help correct diseases that have multiple genetic causes, such as heart disease, obesity, and many cases of autism."

To test the computer's AI design code, Noyes and his team used a customized zinc finger to disrupt the coding sequence of a gene in human cells. In addition, they built several zinc fingers that successfully reprogrammed transcription factors to bind near a target gene sequence and turn up or down its expression, demonstrating that their technology can be used for epigenetic changes.

Noyes, an assistant professor in the Department of Biochemistry and Molecular Pharmacology at NYU Langone, cautions that, while promising, zinc fingers can be difficult to control. Since they are not always specific to a single gene, some combinations can affect DNA sequences beyond a particular target, leading to unintended changes in genetic code.

As a result, Noyes says the team next plans to refine their AI program so it can build more precise zinc-finger groupings that only prompt the desired edit. Noyes is also a member of NYU Langone's Institute for System Genetics.

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CRISPR gene editing reveals biological mechanism behind common blood disorder

UNSW researchers have used CRISPR gene editing -- a type of 'molecular scissors' -- to understand how deletions in one area of the genome can affect the expression of nearby genes. The work, led by UNSW Associate Professor Kate Quinlan and Professor Merlin Crossley, together with collaborators from the US, will help researchers investigate new therapeutic approaches for one of the world's most devastating genetic blood disorders -- sickle cell disease.

Asymptomatic sickle cell disease patients actually lack a tiny part of the genome, scientists have shown.

The team's findings are published today in academic journal Blood. (Just last week, A/Prof. Quinlan and Prof. Crossley received a $412,919 ARC linkage grant to fund a collaboration between UNSW Sydney and CSL that follows on from the work described in this paper.)

"Sickle cell disease and beta thalassemia, a closely related disease, are inherited genetic conditions that affect red blood cells. They are fairly common worldwide -- over 318,000 infants with these conditions are born every year, and haemoglobin disorders cause 3 per cent of deaths in children aged under five years worldwide," says co-lead author A/Prof. Quinlan.

Genetic mutations -- specifically, a defect in the adult globin gene -- are responsible for the disorders. The mutant genes affect the production of haemoglobin, the protein in red blood cells that carries oxygen around our bodies.

"Interestingly, when children are born, they don't show disease symptoms at first, even if they have the mutations, because at that stage, they're still expressing foetal globin and not yet adult globin. That's because we have different haemoglobin genes that we express at different stages of development," says A/Prof. Quinlan.

"As the foetal globin gets turned off, and adult globin gets turned on -- which happens within about the first year of life -- the symptoms start to manifest."

When that happens, the red blood cells take on unusual, sickled shapes and block small blood vessels, causing pain, organ damage, and premature death. The disease is particularly common in tropical countries, and in people from places where malaria is endemic.

"The goal of our research is finding out how we can reverse the foetal to adult globin switch, so that patients continue to express foetal globin throughout life, rather than the mutant adult globin genes that cause blood cells to become stiff and block vessels," says A/Prof. Quinlan.

Interestingly, this already happens in some people with sickle cell disease: thanks to another, beneficial genetic mutation, a rare subset of patients keeps the foetal globin gene 'on' throughout their life and are protected from sickle cell symptoms.

"In these patients, the persistent expression of foetal globin effectively compensates for the defective adult globin -- but up until this piece of research, we didn't really understand the process that led to this incredible advantage," A/Prof. Quinlan says.

'Deleting' genes with CRISPR

To get to the bottom of what's going on in these lucky people's genome, UNSW PhD student Sarah Topfer compiled data on the rare families that express foetal globin throughout life.

"As a first step, Sarah compared deletions in lots of different patients' genomes -- essentially, she looked to see if any shared element was missing in all of them. What do these patients have in common? She found one very small region was deleted in all these patients' genomes."

Sarah then used CRISPR gene editing to replicate some of these big patient deletions -- and the small deleted bit they all had in common -- in cell lines in the lab.

"CRISPR allows us to 'cut' bits of DNA out of cells grown in the lab, to modify genes and see what happens as a result -- it's essentially a tool to figure out what genes do inside living cells," A/Prof. Quinlan says.

"We found that deleting just that one little bit was sufficient to make foetal globin go up and adult globin down -- which suggests that we have found the key mechanism that can explain why foetal globin levels remains high in these asymptomatic patients," A/Prof. Quinlan says.

"Effectively, by deleting the adult globin 'on switch', we made the foetal globin 'on switch' active."

Prof. Quinlan says the results were unexpected.

"It was surprising to see the findings -- many people have studied these mutations for many years, so the idea that there'd be one unifying hypothesis that could explain them rather than them all working through different mechanisms will be surprising for the field.

"While we went in with the hypothesis that there might be one mechanism, we didn't expect it to come out so cleanly -- we thought that perhaps it would be more complicated than what we'd initially thought."

The CRISPR revolution and potential therapies

Co-lead author Prof. Crossley, who is also UNSW's Deputy Vice-Chancellor, Academic & Student Life, says it was impossible to test this model prior to the advent of CRISPR gene editing.

"Our group has specialised in using this new technology to understand globin gene switching," Prof. Crossley says. "Australia now has a significant number of people with either sickle cell disease or thalassemia.

"The work, supported by the National Health and Medical Research Council, is an important example of how the CRISPR gene editing revolution is accelerating scientific understanding and will deliver new therapies to the clinic."

The scientists say the work revealed today is improving our fundamental understanding of the mechanism behind sickle cell disease.

"What this really helps us to do is understand this process of turning off foetal globin and turning on adult globin and how we could reverse that, so that we can use this understanding of the mechanism to help us look for new therapeutic approaches -- it's a key piece of the puzzle," A/Prof. Quinlan says.

Some of Prof. Crossley's team's previous discoveries in the field are informing clinical trials already -- by using beneficial mutations they've discovered in the past that could lead to therapies for these disorders.

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Using gene scissors to specifically eliminate individual cell types

With the help of the CRISPR/Cas molecular scissors, genetic information in a plant can be modified to make the latter more robust to pests, diseases, or extreme climatic conditions. Researchers of Karlsruhe Institute of Technology (KIT) have now developed this method further to eliminate the complete DNA of specific cell types and, thus, prevent their formation during plant development. This will also help scientists better understand development mechanisms in plants. The findings are presented in Nature Communications.

By means of molecular scissors, the DNA -- the carrier of genetic information -- can be modified in plants. So far, the CRISPR/Cas method co-developed in plants by Professor Holger Puchta, molecular biologist at KIT's Botanical Institute has already been used to specifically insert, exchange or combine genes. The goal is to increase the plant's resistance to diseases and environmental impacts. CRISPR (stands for Clustered Regularly Interspaced Short Palindromic Repeats)/Cas are molecular scissors that can specifically recognize and cut DNA sequences. "We have studied molecular scissors for plant use for 30 years now. In the beginning, we applied them to modify individual genes. Two years ago, we were the first worldwide to restructure complete chromosomes," Puchta says. For his research, the pioneer of genome editing twice received the Advanced Grant of the European Research Council (ERC). "We were able to optimize this method. With CRISPR-Kill, we have reached now an entirely new level of development: We can eliminate certain plant cell types and prevent the formation of specific plant organs."

Eliminating Secondary Roots and Petals with CRISPR-Kill

The experiments carried out by the scientists concentrated on secondary roots and petals of the model plant thale cress (Arabidopsis thaliana). "These are classical examples in biology. Here, we know the genetic program and the cell types that are important for the formation of these plant organs," the molecular biologist explains. After the elimination of these cells, CRISPR-Kill plants no longer formed any petals or secondary roots, whereas the control plants exhibited normal growth.

Contrary to other methods that eliminate cells with cytotoxins or laser radiation, CRISPR-Kill induces multiple cuts in the genome. A genome consists of a certain number of chromosomes, on which the individual genes are arranged in fixed order. "So far, CRISPR/Cas has aimed for exactly one location and has cut once or twice to modify a gene or chromosome," Puchta says. "Now, we have reprogrammed our molecular scissors. They no longer address the genomic DNA only once, but aim in the respective cell type for a sequence that is encountered often in the genome and that is essential for the survival of the cell. In this way, many cuts are induced at the same time -- too many for the cell to repair them. The cell will die."

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New findings on the link between CRISPR gene-editing and mutated cancer cells

A protein that protects cells from DNA damage, p53, is activated during gene editing using the CRISPR technique. Consequently, cells with mutated p53 have a survival advantage, which can cause cancer. Researchers at Karolinska Institutet in Sweden have found new links between CRISPR, p53 and other cancer genes that could prevent the accumulation of mutated cells without compromising the gene scissors' effectiveness. The study, published in Cancer Research, can contribute to tomorrow's precision medicine.

Much hope is being pinned on the potential of gene editing using the CRISPR (gene scissors) method as a crucial part in the precision medicine of the future. However, before the method can become hospital routine, several hurdles need to be overcome.

One of these challenges is associated with how cells behave when subjected to DNA damage, which CRISPR gene editing causes in a controlled fashion. Damage to cells activates the protein p53, which acts as the cell's "first aid" response to DNA damage.

It is already known that the technique is less effective when p53 is active; at the same time, however, a lack of p53 can allow cells to start growing uncontrollably and become cancerous. In over half of all cancers the gene for p53 is mutated and thus unable to protect against uncontrolled cell division. It is therefore important to avoid the enrichment (accumulation) of such mutated cells.

Researchers at Karolinska Institutet have now shown that cells with inactivating mutations of the p53 gene gain a survival advantage when subjected to CRISPR and can thus accumulate in a mixed cell population.

The researchers have also identified a network of linked genes with mutations that have a similar effect to p53 mutations, and shown that the transient inhibition of p53 is a possible pharmaceutical strategy for preventing the enrichment of cells with such mutations.

"It can seem contradictory to inhibit p53 in a CRISPR context," says the study's first author Long Jiang, doctoral student at the Department of Medicine, Karolinska Institutet (Solna). "However, some of the literature supports the idea that p53 inhibition can make CRISPR more effective. In our study we show that this can also counteract the enrichment of cells with mutations in p53 and a group of associated genes."

The research has a contributory potential to the future clinical implementation of CRISPR in having identified a network of possible candidate genes that should be carefully controlled for mutations when cells are subjected to the CRISPR technique. Another possible conclusion is that the transient inhibition of p53 could prove a strategy for reducing the enrichment of mutated cells.

The researchers have also studied the DNA-damage response as a possible marker in the development of more precise guide RNA sequences, which are used to show CRISPR where a DNA sequence is to be altered.

"We believe that the up-regulation of genes involved in the DNA damage response can be a sensitive marker for how much unspecific ('off-target') activity a guide RNA has, and can thus help in the selection of 'safer' guide RNAs," says the study's last author Fredrik Wermeling, researcher at the Department of Medicine, Karolinska Institutet (Solna).

The study is largely based on CRISPR, CRISPR screening experiments on isolated cells and analyses of the DepMap database.

The next step of the research is to understand how relevant the described mechanisms are.

"In cell cultures, we see a rapid and pronounced enrichment of cells with p53 mutations when we subject the cells to CRISPR, provided, however, that cells with mutations are there from the start," says Dr. Wermeling. "So we can show that the mechanism exists and factors that affect it, but don't currently know at what level this is a genuine problem, and that's something we want to explore further in more clinic-centered tests."

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Gene editing could render mosquitoes infertile, reducing disease spread

Mosquitoes spread viruses that cause potentially deadly diseases such as Zika, dengue fever and yellow fever. New U.S. Army-funded research uses gene editing to render certain male mosquitoes infertile and slow the spread of these diseases.

Researchers at the Army's Institute for Collaborative Biotechnologies and the University of California Santa Barbara used a gene editing tool known as CRISPR-Cas9 to target a specific gene tied to fertility in male mosquitoes. CRISPR-Cas9 is a genome editing tool that is creating a buzz in the science world, according to yourgenome.org. It is "faster, cheaper and more accurate than previous techniques of editing DNA and has a wide range of potential applications."

Researchers experimented with the Aedes aegypti mosquitoes, which are found in tropical, subtropical and temperate regions throughout the world. The study, published in the Proceedings of the National Academy of Sciences, discerned how a mutation can suppress the fertility of female mosquitoes.

"This is yet one more important and exciting example of how synthetic biology tools are demonstrating unparalleled utility," said Dr. James Burgess, ICB program manager for the U.S. Army Combat Capabilities Development Command, now known as DEVCOM, Army Research Laboratory. "In this case, it's a precision increase from chainsaw to a scalpel leading to the correct biochemical outcome that could substantially reduce the population of a very infectious mosquito."

To manage populations, scientists use a vector-control practice called the sterile insect technique in which they raise a lot of sterile male insects and they then release these males in numbers that overwhelm their wild counterparts. Females that mate with sterile males before finding a fertile one are themselves rendered infertile, thereby decreasing the size of the next generation.

Repeating this technique several times has the potential to crash the population because each generation is smaller than the last; releasing a similar number of sterile males has a stronger effect over time.

The sterile insect technique is effective in managing a number of agricultural pests, including the Mediterranean fruit fly, a crop pest in California. It has also been attempted with Aedes aegypti mosquitoes, but with limited success.

In the past, scientists used chemicals or radiation to sterilize male Aedes aegypti, but the chemicals or radiation impacted the mosquitoes' health to such an extent that they were less successful in mating with females, which undercuts the effectiveness of the sterile insect technique.

The research team wanted to identify a more targeted approach with less collateral damage, mutating a gene in mosquitoes that specifically caused male sterility without otherwise impacting the insects' health.

"When CRISPR/Cas9 came out several years ago it just offered new opportunities to do things that you couldn't do before," said Dr. Craig Montell, distinguished professor at UC Santa Barbara. "So, the time seemed right to for us to start working on Aedes aegypti."

Using gene editing in male Aedes aegypti, researchers found that the mutant male mosquitoes produced no sperm, and unlike in previous efforts, the sterile studs were otherwise completely healthy; however, the team wasn't sure whether sperm, albeit defective sperm from the sterile males, was needed to render female mosquitoes infertile, or whether the transfer of seminal fluid was all it took.

In one experiment, researchers introduced 15 mutant males into a group of 15 females for 24 hours. Then they swapped the males for 15 wild-type males, and left them there.

"Essentially, all of the females remained sterile," Montell said. "This confirmed that males could suppress female fertility without producing sperm."

Next the researchers set out to determine how timing played into the effect. They exposed the females to mutant males for different lengths of time. The scientists noticed little difference after 30 minutes, but female fertility quickly dropped after that. Montell noted that females copulated twice on average, even during the first 10 minutes. This indicated that females have to mate with many sterile males before being rendered infertile themselves.

Combining the females with the males for four hours cut female fertility to 20% of normal levels. After eight hours the numbers began leveling out around 10%.

According to Montell, Aedes aegypti populations could easily bounce back from an 80% drop in fertility. The success of sterile insect technique comes from subsequent, successive releases of sterile males, where each release will be more effective than the last as sterile males account for an ever-growing proportion of the population.

The team plans to continue investigating mosquito mating behaviors and fertility. They are devising a way to maintain stocks of males so they are only sterile in the wild and not in the lab. In addition, they are characterizing male mating behavior to uncover new ways to suppress mosquito populations.

Read more at Science Daily

Move over CRISPR, the Retrons are coming

Researchers have created a new gene editing tool called Retron Library Recombineering (RLR) that can generate up to millions of mutations simultaneously, and 'barcodes' mutant bacterial cells so that the entire pool can be screened at once. It can be used in contexts where CRISPR is toxic or not feasible, and results in better editing rates.

While the CRISPR-Cas9 gene editing system has become the poster child for innovation in synthetic biology, it has some major limitations. CRISPR-Cas9 can be programmed to find and cut specific pieces of DNA, but editing the DNA to create desired mutations requires tricking the cell into using a new piece of DNA to repair the break. This bait-and-switch can be complicated to orchestrate, and can even be toxic to cells because Cas9 often cuts unintended, off-target sites as well.

Alternative gene editing techniques called recombineering instead perform this bait-and-switch by introducing an alternate piece of DNA while a cell is replicating its genome, efficiently creating genetic mutations without breaking DNA. These methods are simple enough that they can be used in many cells at once to create complex pools of mutations for researchers to study. Figuring out what the effects of those mutations are, however, requires that each mutant be isolated, sequenced, and characterized: a time-consuming and impractical task.

Researchers at the Wyss Institute for Biologically Inspired Engineering at Harvard University and Harvard Medical School (HMS) have created a new gene editing tool called Retron Library Recombineering (RLR) that makes this task easier. RLR generates up to millions of mutations simultaneously, and "barcodes" mutant cells so that the entire pool can be screened at once, enabling massive amounts of data to be easily generated and analyzed. The achievement, which has been accomplished in bacterial cells, is described in a recent paper in PNAS.

"RLR enabled us to do something that's impossible to do with CRISPR: we randomly chopped up a bacterial genome, turned those genetic fragments into single-stranded DNA in situ, and used them to screen millions of sequences simultaneously," said co-first author Max Schubert, Ph.D., a postdoc in the lab of Wyss Core Faculty member George Church, Ph.D. "RLR is a simpler, more flexible gene editing tool that can be used for highly multiplexed experiments, which eliminates the toxicity often observed with CRISPR and improves researchers' ability to explore mutations at the genome level."

Retrons: from enigma to engineering tool

Retrons are segments of bacterial DNA that undergo reverse transcription to produce fragments of single-stranded DNA (ssDNA). Retrons' existence has been known for decades, but the function of the ssDNA they produce flummoxed scientists from the 1980s until June 2020, when a team finally figured out that retron ssDNA detects whether a virus has infected the cell, forming part of the bacterial immune system.

While retrons were originally seen as simply a mysterious quirk of bacteria, researchers have become more interested in them over the last few years because they, like CRISPR, could be used for precise and flexible gene editing in bacteria, yeast, and even human cells.

"For a long time, CRISPR was just considered a weird thing that bacteria did, and figuring out how to harness it for genome engineering changed the world. Retrons are another bacterial innovation that might also provide some important advances," said Schubert. His interest in retrons was piqued several years ago because of their ability to produce ssDNA in bacteria -- an attractive feature for use in a gene editing process called oligonucleotide recombineering.

Recombination-based gene editing techniques require integrating ssDNA containing a desired mutation into an organism's DNA, which can be done in one of two ways. Double-stranded DNA can be physically cut (with CRISPR-Cas9, for example) to induce the cell to incorporate the mutant sequence into its genome during the repair process, or the mutant DNA strand and a single-stranded annealing protein (SSAP) can be introduced into a cell that is replicating so that the SSAP incorporates the mutant strand into the daughter cells' DNA.

"We figured that retrons should give us the ability to produce ssDNA within the cells we want to edit rather than trying to force them into the cell from the outside, and without damaging the native DNA, which were both very compelling qualities," said co-first author Daniel Goodman, Ph.D., a former Graduate Research Fellow at the Wyss Institute who is now a Jane Coffin Childs Postdoctoral Fellow at UCSF.

Another attraction of retrons is that their sequences themselves can serve as "barcodes" that identify which individuals within a pool of bacteria have received each retron sequence, enabling dramatically faster, pooled screens of precisely-created mutant strains.

To see if they could actually use retrons to achieve efficient recombineering with retrons, Schubert and his colleagues first created circular plasmids of bacterial DNA that contained antibiotic resistance genes placed within retron sequences, as well as an SSAP gene to enable integration of the retron sequence into the bacterial genome. They inserted these retron plasmids into E. coli bacteria to see if the genes were successfully integrated into their genomes after 20 generations of cell replication. Initially, less than 0.1% of E. coli bearing the retron recombineering system incorporated the desired mutation.

To improve this disappointing initial performance, the team made several genetic tweaks to the bacteria. First, they inactivated the cells' natural mismatch repair machinery, which corrects DNA replication errors and could therefore be "fixing" the desired mutations before they were able to be passed on to the next generation. They also inactivated two bacterial genes that code for exonucleases -- enzymes that destroy free-floating ssDNA. These changes dramatically increased the proportion of bacteria that incorporated the retron sequence, to more than 90% of the population.

Name tags for mutants

Now that they were confident that their retron ssDNA was incorporated into their bacteria's genomes, the team tested whether they could use the retrons as a genetic sequencing "shortcut," enabling many experiments to be performed in a mixture. Because each plasmid had its own unique retron sequence that can function as a "name tag," they reasoned that they should be able to sequence the much shorter retron rather than the whole bacterial genome to determine which mutation the cells had received.

First, the team tested whether RLR could detect known antibiotic resistance mutations in E coli. They found that it could -- retron sequences containing these mutations were present in much greater proportions in their sequencing data compared with other mutations. The team also determined that RLR was sensitive and precise enough to measure small differences in resistance that result from very similar mutations. Crucially, gathering these data by sequencing barcodes from the entire pool of bacteria rather than isolating and sequencing individual mutants, dramatically speeds up the process.

Then, the researchers took RLR one step further to see if it could be used on randomly-fragmented DNA, and find out how many retrons they could use at once. They chopped up the genome of a strain of E. coli highly resistant to another antibiotic, and used those fragments to build a library of tens of millions of genetic sequences contained within retron sequences in plasmids. "The simplicity of RLR really shone in this experiment, because it allowed us to build a much bigger library than what we can currently use with CRISPR, in which we have to synthesize both a guide and a donor DNA sequence to induce each mutation," said Schubert.

This library was then introduced into the RLR-optimized E coli strain for analysis. Once again, the researchers found that retrons conferring antibiotic resistance could be easily identified by the fact that they were enriched relative to others when the pool of bacteria was sequenced.

"Being able to analyze pooled, barcoded mutant libraries with RLR enables millions of experiments to be performed simultaneously, allowing us to observe the effects of mutations across the genome, as well as how those mutations might interact with each other," said senior author George Church, who leads the Wyss Institute's Synthetic Biology Focus Area and is also a Professor of Genetics at HMS. "This work helps establish a road map toward using RLR in other genetic systems, which opens up many exciting possibilities for future genetic research."

Another feature that distinguishes RLR from CRISPR is that the proportion of bacteria that successfully integrate a desired mutation into their genome increases over time as the bacteria replicate, whereas CRISPR's "one shot" method tends to either succeed or fail on the first try. RLR could potentially be combined with CRISPR to improve its editing performance, or could be used as an alternative in the many systems in which CRISPR is toxic.

More work remains to be done on RLR to improve and standardize editing rate, but excitement is growing about this new tool. RLR's simple, streamlined nature could enable the study of how multiple mutations interact with each other, and the generation of a large number of data points that could enable the use of machine learning to predict further mutational effects.

"This new synthetic biology tool brings genome engineering to an even higher levels of throughput, which will undoubtedly lead to new, exciting, and unexpected innovations," said Don Ingber, M.D., Ph.D., the Wyss Institute's Founding Director. Ingber is also the Judah Folkman Professor of Vascular Biology at HMS and Boston Children's Hospital, and Professor of Bioengineering at the Harvard John A. Paulson School of Engineering and Applied Sciences.

Read more at Science Daily

CRISPR meets Pac-Man: New DNA cut-and-paste tool enables bigger gene edits

 Gene editing for the development of new treatments, and for studying disease as well as normal function in humans and other organisms, may advance more quickly with a new tool for cutting larger pieces of DNA out of a cell's genome, according to a new study by UC San Francisco scientists.

Publication of the UCSF study on Oct. 19, 2020 in the journal Nature Methods comes less than two weeks after two researchers who first used the genetic scissors known as CRISPR-Cas9 were selected to receive this year's Nobel Prize in Chemistry.

Though now employed as a research tool in laboratories around the world, CRISPR evolved eons ago in bacteria as a means to fight their ancient nemeses, a whole host of viruses known as bacteriophages. When bacteria encounter a phage, they incorporate a bit of the viral DNA into their own DNA, and it then serves as a template to make RNA that binds to the corresponding viral DNA in the phage itself. The CRISPR enzymes then target, disable and kill the phage.

In his latest work exploring this ancient and strange arms race, principal investigator Joseph Bondy-Denomy, PhD, associate professor in the UCSF Department of Microbiology and Immunology, joined scientists Bálint Csörg?, PhD, and Lina León to develop and test a new CRISPR tool.

The already renowned CRISPR-Cas9 ensemble is like a molecular chisel that can be used to rapidly and precisely excise a small bit of DNA at a targeted site. Other methods can then be used to insert new DNA. But the new CRISPR-Cas3 system adapted by the UCSF scientists employs a different bacterial immune system. The key enzyme in this system, Cas3, acts more like a molecular wood chipper to remove much longer stretches of DNA quickly and accurately.

"Cas3 is like Cas9 with a motor -- after finding its specific DNA target, it runs on DNA and chews it up like a Pac-Man," Bondy-Denomy said.

This new capability to delete or replace long stretches of DNA will enable researchers to more efficiently assess the importance of genomic regions that contain DNA sequences of indeterminate function, according to Bondy-Denomy, an important consideration in understanding humans and the pathogens that plague them.

"Previously, there was no easy and reliable way to delete very large regions of DNA in bacteria for research or therapeutic purposes," he said. "Now, instead of making 100 different small DNA deletions we can just make one deletion and ask, 'What changed?'"

Because bacteria and other types of cells are commonly used to produce small molecule or protein-based pharmaceuticals, CRISPR-Cas3 will enable biotechnology industry scientists to more easily remove potentially pathogenic or useless DNA from these cells, according to Bondy-Denomy.

"Large swathes of bacterial DNA are poorly understood, with unknown functions that in some cases are not necessary for survival," Bondy-Denomy said. "In addition, bacterial DNA contains large stretches of DNA imported from other sources, which can cause disease in the bacterium's human host, or divert bacterial metabolism."

CRISPR-Cas3 also should also allow entire genes to be inserted into the genome in industrial, agricultural or even in human gene therapy applications, Bondy-Denomy said.

The UCSF researchers selected and modified the CRISPR-Cas3 system used by the bacterium Pseudomonas aeruginosa, and demonstrated in this species and in three others, including bacteria that cause disease in humans and plants, that their more compact version functions well to remove selected DNA in all four species. Other CRISPR-Cas3 systems have been made to work in human and other mammalian cells, and that also should be achievable for the modified P. aeruginosa system, Bondy-Denomy said.

Bondy-Denomy studies a range of bacteria, bacteriophage, and CRISPR systems to learn more about how they work and to find useful molecular tools. "CRISPR-Cas3 is by far the most common CRISPR system in nature," he said. "About 10 times as many bacterial species use a Cas3 system as use a Cas9 system. It may be that Cas3 is a better bacterial immune system because it shreds phage DNA."

Unlike Cas9, when Cas3 binds to its precise DNA target it begins chewing up one strand of the double-stranded DNA in both directions, leaving a single strand exposed. The deletions obtained in the UCSF experiments ranged in size, in many cases encompassing as many as 100 bacterial genes. The CRISPR-Cas3 mechanism should also allow for easier replacement of deleted DNA with a new DNA sequence, the researchers found.

For DNA deletion and editing in the lab, scientists program CRISPR systems to target specific DNA in the genome of an organism of interest using any guide sequence they choose.

In the new CRISPR-Cas3 study, by manipulating the sequences of DNA provided to the bacteria for repairing the deletions, the researchers were able to precisely set the boundaries of these large DNA repairs, something they were unable to accomplish with CRISPR-Cas9.

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New genetic editing powers discovered in squid

Squid

Revealing yet another super-power in the skillful squid, scientists have discovered that squid massively edit their own genetic instructions not only within the nucleus of their neurons, but also within the axon -- the long, slender neural projections that transmit electrical impulses to other neurons. This is the first time that edits to genetic information have been observed outside of the nucleus of an animal cell.

The study, led by Isabel C. Vallecillo-Viejo and Joshua Rosenthal at the Marine Biological Laboratory (MBL), Woods Hole, is published this week in Nucleic Acids Research.

The discovery provides another jolt to the "central dogma" of molecular biology, which states that genetic information is passed faithfully from DNA to messenger RNA to the synthesis of proteins. In 2015, Rosenthal and colleagues discovered that squid "edit" their messenger RNA instructions to an extraordinary degree -- orders of magnitude more than humans do -- allowing them to fine-tune the type of proteins that will be produced in the nervous system.

"But we thought all the RNA editing happened in in the nucleus, and then the modified messenger RNAs are exported out to the cell," says Rosenthal, senior author on the present study. "Now we are showing that squid can modify the RNAs out in the periphery of the cell. That means, theoretically, they can modify protein function to meet the localized demands of the cell. That gives them a lot of latitude to tailor the genetic information, as needed." The team also showed that messenger RNAs are edited in the nerve cell's axon at much higher rates than in the nucleus.

In humans, axon dysfunction is associated with many neurological disorders. Insights from the present study could accelerate the efforts of biotech companies that seek to harness this natural RNA editing process in humans for therapeutic benefit.

Scientists from Tel Aviv University and The University of California at Denver collaborated with MBL scientists on the study.

Previously, Rosenthal and colleagues showed that octopus and cuttlefish also rely heavily on mRNA editing to diversify the proteins they can produce in the nervous system. Together with squid, these animals are known for strikingly sophisticated behaviors, relative to other invertebrates.

From Science Daily

Scientists boost gene-editing tools to new heights in human stem cells

During the past decade, the gene editing tool CRISPR has transformed biology and opened up hopeful avenues to correct deadly inherited diseases. Last fall, scientists began the first human clinical trials using CRISPR to combat diseases like cancer. They remove some of a person's cells, CRISPR edit the DNA, and then inject the cells back in, where hopefully, they will cure the disease.

But along with this promise of regenerative, personalized medicine, CRISPR can also have significant safety limitations. CRISPR may not edit in the right place (so-called off-target gene effects) or not being terribly efficient (successful editing may only be achieved in about 10% of the time for every available cell target).

These limitations have frustrated scientists such as Arizona State University's David Brafman, a cell bioengineer. Brafman initial hopes are to use gene editing to get at the heart of uncovering the causes of studies in his lab of neurodegenerative diseases like Alzheimer's.

"We study neurodegenerative diseases like Alzheimer's and use stem cells to study specific mutations or risk factors associated with Alzheimer's disease," said Brafman, a biomedical engineering faculty member in ASU's Ira A. Fulton Schools of Engineering. "We are not necessarily a gene-editing tool development lab, but we were running into difficulty generating stem cell lines by using a traditional CRISPR-based editing approach. For reasons that are still unknown, stem cells are really resistant to that sort of genetic modification."

Green light means go

Now, Brafman, using a new update to the CRISPR base editing technology originally developed in the lab of David Liu at Harvard, has vastly outperformed previous efforts by making highly accurate, single DNA base editing with an efficiency of up to 90% of human stem cells. The results were published in the journal Stem Cell Reports.

"Previously, with CRISPR, it's just been a random guess," said Brafman. "And so, if you are picking at random stem cells and the efficiency is low, you'll likely get only 10% or 5% because you have no idea if the edits have been made -- the cell isn't telling you."

Brafman's lab has developed a new TREE method (an acronym short for transient reporter for editing enrichment, or TREE), which allows for bulk enrichment of DNA base-edited cell populations -- -and for the first time, high efficiency in human stem cell lines.

""Most of the studies are done in immortalized cell lines or cancer cell lines, which are relatively easy to edit," said Brafman. "This is the first example of using base editors in pluripotent stem cells, which is a very valuable cell population to genetically modify. We envision this method will have important implications for the use of human stem cell lines in developmental biology, disease modeling, drug screening and tissue engineering applications,"

Last year, they had shown that their TREE approach can work in human cell lines, but wanted to further push the technology further to find a way to rapidly and efficiently edit human stem cell lines.

Unlike CRISPR, which cuts across both DNA stands, their TREE method only makes a single strand nick in DNA. For example, when a single DNA base is successfully edited from a C to a T, a protein gives off a signal, turning from blue to green.

"Now, if a cell is telling you, 'if I'm glowing green I have a 90% chance of being edited you are going to have better luck identifying edited populations," said Brafman. "Then, you can exclude all of the cells that are not edited. We isolate single cells that are glowing green, then grow those up into clonal populations that you are able to expand indefinitely."

Targeting Alzheimer's

Pluripotent stem cells are valued for regenerative medicine because they have the ability to become or differentiate into any cell type in the human body.

Brafman explains that there are two general sources, "embryonic stem cells, which are derived from the inner cell mass of a preimplantation blastocyst, and then there are induced pluripotent stem cells, which are derived from taking somatic cells like skin or blood from patients."

Brafman's lab uses the induced pluripotent stem cells for their research.

"For this study, we used pluripotent stem cells from both healthy patients and then patients with Alzheimer's disease. Some of the genes that we were interested in modulating are related to Alzheimer's disease. The majority of the patients suffering from Alzheimer's disease suffer from late onset, or sporadic Alzheimer's disease."

To provide their proof-of-concept, they targeted the APOE gene, which can come in three flavors. One of the three gene variants, called APOE4, has been associated with a higher risk for late onset Alzheimer's disease. For the study, they introduced single DNA based edits into the APOE gene.

"That's why we are interested in having these cells," said Brafman. "They are representative of the neurons and the various cell types in the central nervous system with patients with these various risk factors. Then, we can understand why an APOE variant can increase or decrease risk, and then we can start targeting those pathways that are affected."

Not only could TREE make single DNA edits to the APOE4 gene, but unlike CRISPR, make highly accurate corrections to both copies of the APOE4 gene that humans possess.

"The traditional CRISPR approach is that you have to edit once to get a heterozygous edit , then isolate that clone, edit again to get another heterozygous edit," said Brafman. "So, it's very inefficient in that way. We are generating homozygous edits at an efficiency approaching 90%. I haven't seen any other technologies that can do that in pluripotent stem cells."

In addition, TREE could also be used to engineer critical gene knockout mutations into stem cell lines. "The most fundamental experiment you can do if a gene has important implications in disease, development or physiology is knock it out," said Brafman. That opens up a whole bunch of questions that we can address. Using APOE as a case study, now we can knock out APOE in these cells if you don't have APOE at all. Is it beneficial? Detrimental? Or no difference?"

Complex cases

While diseases like sickle-cell anemia or cystric fibrosis are caused by single mutations in DNA, for most diseases and leading causes of death, like heart disease or high blood pressure, are complex, and involve multiple genes. Brafman wanted to also address the complex, root causes of Alzheimer's.

"Especially as it related to Alzheimer's disease, there can be multiple risk factors that act in concert, so we wanted a way to introduce multiple edits simultaneously in pluripotent stem cells. Because otherwise, you would have to take this sequential iterative approach, where you introduce one edit, isolate a clonal population introduce another edit, and so on.

They successfully demonstrated that TREE could be used to make new stem cell lines that had been simultaneously edited at multiple gene locations. Their results showed that more than 80% of stem clones had been targeted at all three different gene sites, and with all clones editing both gene copies.

"We found that if you multiplex you still get the same efficiency of editing as you would if you just edited a single allele," said Brafman. "Now, we can use these cells as in vitro models to study the disease and screen drugs."

Brafman is hopeful that their new tools will generate excitement in the gene editing community, and spur others on to make new discoveries.

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