Future of Genome Editing - Looking Beyond CRISPR



Genome editing (GE) has revolutionized biological research through its ability to precisely edit the complete set of genes or genetic material present in a cell of living organisms, including humans, plants, animals, and microbes. In recent years, various GE tools have been explored to edit both simple and complex genomes. It can be used either to add desirable or remove undesirable alleles simultaneously in a single event.


In less than a few years, the gene-editing technology Clustered Regularly Interspaced Short Palindromic Repeats commonly known as known CRISPR has revolutionized modern biology's face and generated excitement for new and improved gene therapies. It is dramatically more manageable, cheaper, and more versatile than previous technologies and enables scientists to alter genomes practically at will. It's not perfect, but it has already been used to correct genetic diseases in humans and animals. The problem with this approach is that it only works for certain types of DNA. For example, if you had a disease called "Leber's Hereditary Optic Neuropathy" and your eyesight was starting to go, you could theoretically use CRISPR to correct the defective gene that causes it, but you'd still get the other problems caused by the mutation you got first (in this case, bad vision). This is one of the limitations.


With other diseases, the situation is slightly better, but not much. Editing out diseases is possible, but it's still in the very early testing stages. The other big limitation of CRISPR is that it's hard to ensure a precise cut. This means if multiple possible genes could be cut, many of the edited cells may have the wrong change, which leads to messed up cells and tissues.


Usually, when referring to CRISPR, it means Crispr/Cas9 - a riboprotein complex composed of a short strand of RNA and an efficient DNA-cutting enzyme. It has high efficiency, accuracy, and ease of use. Newly emerging CRISPR/Cas systems like spCas9-NG, base editing, xCas9, Cpf1, Cas13, and Cas14 are now being used for GE.


So, the question facing researchers using these technologies is: “what is going to happen when the scientist makes the change? Sure, he will get the expected change but what else, unexpected, is going to happen?” Scientists are looking at ways to answer this tricky question and are experimenting with Artificial Intelligence. Microsoft has built a machine learning tool to help in CRISPR/Cas9 design and has developed two predictive modeling approaches, Azimuth and Elevation, to tackle the problems of on-target and off-target activity prediction.


Using an in-depth learning approach, the tool can discover the correlation between the editing applied to the genome and the consequences at a global scale, basically linking the genotype with the phenotype. In the future, this may provide a crucial tool to fix problems appearing at the phenotype level by tweaking with the genome (it will also show that some issues do not have a solution with this approach, since fixing something -like changing the number of fingers in a hand- will ruin something else).


Many startups are making their presence felt in the arena of genome engineering. Inscripta launched the world’s first benchtop platform for digital genome engineering. They have an enzyme engineering program that allows their customers to create their own customized gene-editing applications. Inscripta has also created a family of CRISPR enzymes (MADzymes), which have innovative features, said to increase the speed and efficiency of precision genome editing.


Inari Agriculture is building the world's first ‘Seed Foundry’ through which they are reintroducing nature's genetic diversity and working to address some of today's significant challenges, including climate change. They’re focusing on using CRISPR to manage specific gene expressions in plants. They are also developing customized seeds that significantly reduce the land, water, and other natural resources required to produce food and feed. These CRISPR-edited seeds are planting a sustainable future for the agricultural industry.


Synthego, the genome engineering company, is designing new foundational technology for standardized precision and control of CRISPR-based gene editing inside cells. They have come up with a CRISPR Design Tool, which uses several built-in algorithms to identify guide sequences targeting a gene, and simplifies gRNA design using light. They’re also developing a Gene knockout kit v2 that’s designed to guarantee a gene knockout, saving scientists from trial and error cycles in their CRISPR experiments.


CRISPRomics is an industrialized discovery engine of KSQ Therapeutics that utilizes a suite of proprietary CRISPR/Cas9 tools to generate disease-specific insights for every human gene with improved precision and at an unprecedented scale. They have evolved this engine into multiple distinct platforms to identify and genetically validate optimal novel targets for drug discovery. CRISPRomics has broad utility across numerous therapeutic areas, and the company is currently deploying this approach in oncology, immuno-oncology, autoimmune disease, and select rare diseases.


eGenesis are leaders in gene editing and genome engineering and are uniquely positioned to address the organ crisis with their multiplexed gene-editing platform. They use rapid and automated DNA sequencing to perform microbial fingerprinting and have been awarded the first US Patent for their fast DNA sequencing platform to control hospital-acquired infections.


eGenesis has partnered with Quihan Bio (China) in using CRISPR to create the most extensively genetically engineered pigs, whose tissues have all the features necessary for being transplanted into humans.


Verve Therapeutics, a biotech company pioneering gene-editing medicines to treat cardiovascular disease, is developing one-time gene editing medicines to safely and precisely turn off a gene in the liver to permanently lower LDL cholesterol or triglyceride levels and thereby treat adults with coronary heart disease, the leading cause of death worldwide. They target base editing to knock out PCSK9 or ANGPTL3 in the liver and substantially reduce blood levels of LDL cholesterol or triglycerides. Coronary heart disease occurs when cholesterol-laden plaque builds up in the heart's arteries, which can restrict blood flow and lead to a heart attack.


Tango Therapeutics is leveraging the principle of synthetic lethality to develop medicines that take direct aim at specific tumors. Using an approach that starts and ends with patients, they’re expanding the reach of genetically targeted therapies. They have built a target discovery platform that uses CRISPR to find vulnerabilities in specific cancers. Tango Therapeutics collaborated with Gilead Sciences in 2018 to discover, develop, and commercialize a pipeline of innovative targeted immuno-oncology therapies.


Beyond CRISPR


A mini-me: The components of CRISPR–Cas9 system - Cas9 and a strand of RNA- are too large to stuff into the virus's genome, most commonly used in gene therapy to shuttle foreign genetic material into human cells. A solution comes in the form of a mini-Cas9, which was plucked from the bacterium Staphylococcus aureus. It’s small enough to squeeze into the virus used in one of the gene therapies currently on the market. Two groups used the mini-me Cas9 in mice to correct the gene responsible for Duchenne muscular dystrophy.


Expanded reach: Cas9 will not cut everywhere it’s directed to. A specific DNA sequence must be nearby for that to happen. This demand is easily met in many genomes but can be a painful limitation for some experiments. It has been surpassed by one such enzyme, called Cpf1, smaller than Cas9, it has different sequence requirements and is highly specific. Another enzyme, called C2c2, targets RNA rather than DNA - a feature that holds the potential for studying RNA and combating viruses with RNA genomes.


True editors: Many labs use CRISPR–Cas9 only to delete sections in a gene, thereby abolishing its function. Those who want to swap one sequence with another face a more difficult task. When Cas9 cuts DNA, the cell often makes mistakes as it stitches together the broken ends. This creates the deletions that many researchers desire. But researchers who want to rewrite a DNA sequence rely on a different repair pathway that can insert a new sequence — a process that occurs at a much lower frequency than the error-prone stitching. Researchers announced that they had disabled Cas9 and tethered to it an enzyme that converts one DNA letter to another. The disabled Cas9 still targeted the sequence dictated by its guide RNA but could not cut: instead, the attached enzyme switched the DNA letters, ultimately yielding a T where once there was a C.


Pursuing Argonautes: Researchers claimed that they could use a protein called NgAgo to slice DNA at a predetermined site without needing a guide RNA or a specific neighboring genome sequence. Instead, the protein — which is made by a bacterium — is programmed using a short DNA sequence that corresponds to the target area.


What’s next?


Genome-editing technology applies for the good of humankind and the planet. The genome-editing wish list includes better methods for multiplexing-editing more than one gene at a time. Given its popularity and availability, CRISPR dominates genome-editing predictions. CRISPR-based systems will continue to improve incrementally. CRISPR is already very powerful, and so many people are working on it and other genome-editing systems that they'll inevitably continue to improve.


The full realization of the potential of CRISPR/Cas9 approaches will require addressing many challenges. It is somewhat clunky, unreliable, and a bit dangerous too. It can't bind to just any place in the genome. It sometimes cuts in the wrong places, and it has no off-switch. Cas9 is large, so its gene is challenging to deliver to cells via vectors such as adeno-associated viruses commonly used in gene therapy. Also, scientists worry about off-target effects. This remains the most significant obstacle for CRISPR/Cas9 use regarding gene cargo delivery systems, and an all-purpose delivery method has yet to emerge. Instead, multiple ways are seen for delivering CRISPR to cells. Every technique has both advantages and disadvantages, and some can be quite specific or ill-suited to certain types of delivery.


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