Gene Editing: A Primer

In 1953, the discovery of DNA’s structure and function galvanized innovation in genetics—the study of genes, genetic variation, and heredity. Yet, many foundational concepts in genetics, gene sequencing, and gene editing have been difficult to grasp, until recently (Pray 2008).

In hopes of curing disease, Emmanuelle Charpentier and Jennifer Doudna developed a revolutionary gene-editing method called CRISPR/Cas9 and won the Nobel Prize in Chemistry in 2020 (NobelPrize.org 2020). Given widespread curiosity around CRISPR/Cas9, its mechanism, and its applications, this Primer offers readers an overview of gene editing.

After a brief history reviewing core concepts, we will delve into the different gene-editing techniques.

Historical Overview[1]

During the mid-nineteenth century, Charles Darwin and Alfred Russel Wallace shocked the scientific world with a revolutionary idea that species are a product of natural selection and evolution. Their hypothesis begged questions. How do traits pass from parents to offspring? What processes underlie the expression of traits explaining both diversity and homogeneity?

Gregor Mendel’s experiments on pea plants answered some questions, in particular that traits like plant height and color varied according to parent traits. Somewhat misunderstood during his lifetime, Mendel’s work regained prominence when biologist William Bateson coined the term “genetics” in the study of inheritance (Bateson 1905). Before Mendel’s work, most scientists assumed that offspring traits were an average of parent traits (Bulmer 1999). Yet, if so, each trait would have converged to its average after multiple generations. In other words, the “average” theory did not explain diversity within and across species. In contrast, Mendel’s laws of inheritance suggested that offspring traits were a function of discrete contributions, one from each parent.

Mendel’s laws did not answer other questions about heredity In 1869, Friedrich Miescher isolated nucleic acid, one of the primary building blocks of DNA, but not until 1944, when Oswald Avery, Colin MacLeod, and McCarty manipulated the cell contents of bacteria, did proof evolve that DNA was the repository of hereditary information (Pray 2008). Even before discovering DNA’s role in heredity, scientists understood that cells exchange genetic material during both somatic (non-sex) and sex cell reproduction (Cohen 2018).

Identifying DNA as the hereditary content governing biological systems was the first step in understanding the function of genetic code. In the 1950s, James Watson, Francis Crick, Rosalind Franklin, and Maurice Wilkins revealed DNA at the molecular level, enabling scientists to progress toward gene sequencing and editing.

DNA sequences encode for the synthesis of specific proteins that govern bodily processes, a relationship called the Central Dogma. The Central Dogma, in turn, has raised more questions.

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How might we manipulate DNA sequences to alter biological processes or functions?

Scientific Progress in Genetics[2]

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Core Concepts[3]

What are the fundamental molecules that govern biological systems? This section illustrates them in order of physical size.

Cells: Cells are the smallest living units in our bodies (National Cancer Institute 2011). Our bodies house highly specialized living cells. Skin and blood, for example, are made up of trillions of cells that are differentiated to serve distinct functions.

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Each of our ~200 cell types—from stem cells to blood cells, to nerve cells, to fat cells—has a miniature, organ-like structure called the nucleus that contains hereditary information within chromosomes (Baxter 2021).

Chromosomes: Our hereditary information is highly complex and organized into structures called chromosomes. Inside the nucleus of each human cell are 23 pairs of chromosomes, or 46 individual structures.[4] The number of chromosomes varies from species to species. Bacteria often have single DNA molecules, while strawberries can have 56 or even 70 total chromosomes per cell (Hummer et al. 2009).

Genes: Each chromosome is like a marketplace with different stores or sections called genes. Genes often contain instructions that synthesize proteins, but some have no use at all. The genome is the complete set of genes or genetic information, but only 1-2% of the human genome contains protein-coding genes. In other words, ~99% of the “stores” in the “shopping center” are “unoccupied” or non-functional (Henninger 2012). Scientists estimate that humans have ~20,000 genes.

Gene activation is fundamental to the development and functioning of all living organisms, simultaneously enabling diversity and homogeneity. While a human’s appearance, health, and personality can differ significantly from her friend’s, for example, the two friends’ genomes have much more in common with each other than with elephants.

While one gene can determine one trait—Huntington’s disease, for example (Bhattacharyya 2008)—multiple genes and the environment determine most traits. A single gene also can influence multiple traits (Lobo 2008).

Proteins: How do genes determine traits? Technically, they hold the instructions that encode proteins. Proteins are organic compounds made up of building blocks called amino acids that serve specific and vital roles in the growth and maintenance of our cells (EUFIC 2019).

Chemical Structure Depiction

Proteins are the primary workforce in the human body: they control most of its structures, execute chemical reactions, and process signals and information (Ahlgren 2021). Without them, life would cease to function.

DNA: To understand DNA, chromosomes are necessary. Chromosomes make up DNA and other molecules that hold the DNA together. Genes are specific regions that encode proteins based on DNA-enabled instructions. DNA is made up of two strands connected in a ladder-like structure that, in turn, is twisted into a double helix.

DNA Bases: Each rung of the DNA ladder is made of two molecules joined together by hydrogen bonds. DNA comprises four primary molecules: adenine, thymine, cytosine, and guanine. Adenine always pairs with thymine, and cytosine always pairs with guanine. These molecules (or “bases”), and the order in which they appear, provide the genetic code—the instructions—necessary for cells to organize various amino acids. When joined, amino acids make up a specific protein. Together, they power life.

Structure of DNA

RNA: In humans, RNA is a single-strand compound that sends messages between DNA and amino acids (Clancy and Brown 2008). To understand the process, consider this analogy. Imagine a teacher who speaks only English, and all her students speak only Spanish. The English-speaking teacher wants her students to line up single file in alphabetical order according to their last names. Fortunately, her Spanish-speaking friend can translate the request. In this example, the English-speaking teacher is analogous to DNA and her Spanish-speaking friend, RNA, while each child is analogous to an amino acid, and the entire line of children, the protein.

Nucleic Acid Strand

Gene Editing: A Primer

What Is The Potential Of Gene Editing?

Gene editing has the potential to cure a range of life-threatening genetic disorders. Take the example of cystic fibrosis. What is it, and how might gene editing help those who have it?

1. Let’s start with a simple illustration. At a point on a specific chromosome in cells, most people have a DNA sequence like this one:

2. We then identify a person with this DNA sequence:

For most people in our hypothetical universe, the AAA DNA sequence codes for a specific amino acid called phenylalanine. For the unique person, the AAT DNA sequence codes for an amino acid called leucine. With trillions of cells continuously reproducing and synthesizing proteins, this simple error can cascade into devastating consequences (Drummond and Wilke 2009).

3. Now, consider that the DNA sequence in question is in a region called the CFTR gene, which is responsible for making a protein called the cystic fibrosis transmembrane conductance regulator. That regulator facilitates the transport of particles through the walls of cells that produce mucus, sweat, and other biological substances (Winikates 2012). While the AAA DNA sequence produces phenylalanine to synthesize the CFTR protein, the AAT sequence produces leucine—incorrectly, so to speak, because, without phenylalanine, the CFTR gene cannot produce the CFTR protein properly, leading to cystic fibrosis (Canadian Agency for Drugs and Technologies in Health 2018).

4. We know that something in the AAA DNA is giving rise to cystic fibrosis. What if we could target the mutant section of AAA DNA in the group of cells, make the required changes, allow the cells to divide, and replace all the error-ridden cells with error-free cells? In other words, what if we could edit the DNA, so that it evolves correctly instead of creating cystic fibrosis?

This simplified, hypothetical problem prepares us to delve into the different gene-editing techniques and some of their applications.

What Is Gene Editing?

Genetic engineering took hold in the 1970s when Rudolf Jaenisch and Beatrice Mintz injected viral DNA into the embryos of mice (Jaenisch and Mintz 1974). Many scientists experimented further, introducing foreign hereditary materials into test subjects and, in the 1980s, three groups of scientists developed a method of targeting and deactivating specific mice genes (NobelPrize.org 2007).

The method relied on a process called homologous recombination, in which similar chromosomes exchange DNA sequences—or RNA, for viruses—with one another, increasing genetic variation in the process (Masood et al. 2016). Scientists found that a similar interaction can occur between foreign DNA and the chromosomes of mammal cells (NobelPrize.org 2007). The method showed that the elimination of diseases caused by mutations in the genome was possible. Scientists then began to evolve more efficient ways of targeting genes (Woolf 1998).[5]

Methods of Gene Editing: Meganucleases

Meganucleases are enzymes that recognize DNA sequences ranging from 12 to 45 base pairs and can cut the DNA at specific sites. In the 1990s, scientists discovered that these enzymes could be important for genetic engineering (Epinat et al. 2003), acting like “scissors” that create breaks in specific DNA locations to replace the sequence entirely or to insert new bases within the break.

Because meganucleases target large sequences, their applications are limited since the chance of finding large, specific sequences in a gene is low. As a result, scientists are evolving and expanding the library of sequences that meganucleases can target—either by modifying the targeted DNA sequences or by creating artificial meganucleases (Hoy 2013).

An Illustration Of Genetic Engineering via Meganucleases

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Because of their highly specific nature, the work with meganucleases is difficult and costly; but that has not halted their development. Currently, biotechnology company Precision Biosciences is conducting a Phase 1 clinical trial on the efficacy of specially engineered meganucleases to target non-Hodgkin lymphoma (O’Hanlon Cohrt 2021). Recognizing specific DNA sequences, meganucleases have the advantage of low cell toxicity: the risk that they cut the DNA at off-target or incorrect locations is low compared to other gene-editing techniques (Paques and Duchateau 2007).

Methods of Gene Editing: Zinc Finger Nucleases (ZFNs)

While meganucleases cut DNA at highly specific sites, Zinc Finger Nucleases can target up to 18 base pairs specified by scientists using Lego-like structures called zinc fingers (Martinez-Lage et al. 2017). Zinc fingers are natural protein structures that bind to different DNA sequences (Klug 2010).

Each zinc finger can recognize DNA sequences of three or four base pairs and, when combined, they can identify and bind to a specific DNA sequence 24 base pairs or longer in length (Kim et al. 2009). Typically, zinc fingers are more flexible than meganucleases in genetic engineering applications. Combining zinc fingers with a DNA-cutting enzyme similar to but less specific than meganucleases, scientists have created ZFNs that target and cut a wide range of DNA with unprecedented precision.

That said, ZFNs are not risk free. If zinc fingers are not specific enough for the target DNA sequence, the nuclease can cut DNA unintentionally at undesired, off-target locations, potentially killing the cell altogether (Cornu et al. 2008). While the search for zinc fingers sufficiently specific to the targeted DNA sequence has been difficult, recent investment in enhanced zinc finger engineering techniques is addressing those issues (Paschon et al. 2019).

In 2021, a group of researchers from California and Australia published promising results from a study exploring the efficacy of zinc finger proteins in repressing the expression of HIV-1, as opposed to forcing HIV-positive patients into stressful therapy sessions (Shrivastava et al. 2021). In addition, Sangamo Therapeutics, which is developing ZFN-based gene-editing techniques to treat sickle cell disease, released positive Phase I/II results in three patients undergoing gene-edited cell therapy for sickle cell disease (Philippidis 2021).

An Illustration Of Genetic Engineering via ZFNs

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Methods of Gene Editing: Transcription Activator-Like Effector Nucleases (TALENs)

With one key difference, TALENs are similar to ZFNs: whereas each zinc finger recognizes multiple bases—typically three—on a target sequence, each Transcription Activator-Like Effector (TALE) protein recognizes only one base. As a result, scientists can create structures that identify specific sequences in the genome (Yeadon 2014). In 2010, researchers from Iowa State University, developed the method of combining TALE proteins with DNA-cutting enzymes and created a more flexible alternative to ZFNs (Christian et al. 2010).

Since their initial discovery, TALENs have been used extensively to modify the genomes of various plants. In 2014, a group of researchers used TALENs to introduce mutations into two genes in soybeans, causing their seeds to convert mono-unsaturated fat into poly-unsaturated fat for healthier for healthier soybeans in human diets (Haun et al. 2014). Since its founding in 2010, agricultural technology company Calyxt has leveraged TALENs and currently is developing oats that can grow in cold climates (Calyxt 2021).

TALENs also are being developed for delivery in humans. Biotech company Cellectis, for example, uses TALENs to edit the genes of donor-derived white blood cells (T-cells) and inserts them into patients with leukemia and multiple myeloma (Cellectis 2021).

An Illustration Of Genetic Engineering With TALENs

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Methods of Gene Editing: CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) + Cas9 Proteins

Unlike the techniques discussed above, CRISPR/Cas9 does not require scientists to spend time constructing new protein structures to target a specific DNA sequence. Rather, they can program an answer-key—the single guide RNA (sgRNA)—to target a specific DNA sequence. When combined with a DNA cutting protein called Cas9, CRISPR/Cas9 enables the most efficient and flexible gene editing. While CRISPR Cas9 was the first enzyme to be discovered and researched, other Cas enzymes have different properties.

“Tandem repeats” in DNA refer to repeated, recognizable patterns in DNA bases that are adjacent to each other (National Human Genome Research Institute 2021). The sequences “CATCATCATCAT” and “AGAGAGAGAGAG” are tandem repeats, for example. During the 1980s, scientists discovered repeats in the genome of the bacteria E. coli which, unlike most tandem repeats, were separated by spacer sequences, or segments of DNA that separate two active genes from each other (Vidyasagar and Lanse 2021). These sequences also are palindromic, meaning that the sequence of one part of the ladder is identical to the sequence on the other side of the ladder in reverse.

Research on these mysterious repeat sequences has revealed (1) that these “repeats” are present in many other bacteria and archaebacteria, and (2) that these sequences have some association with other genes that produce DNA-cutting proteins (Mojica et al. 2000). By 2007, researchers conceptualized the function of CRISPR in bacteria as a memory bank for previous attacks from foreign viruses (Barrangou et al. 2007). After a viral attack, the bacteria would incorporate parts of the viral genome into its own DNA as spacer sequences so that, in future attacks, the bacteria could direct the DNA-splicing proteins (Cas9 proteins)—with the help of CRISPR RNA (crRNA) and trans-activating RNA (tracrRNA)—to cut parts of the virus genome using the sequences the bacteria already had stored (Deltcheva et al. 2011; Jinek et al. 2012; Vidyasagar and Lanse 2021).

These bacteria act like federal judges. The first time the virus commits a crime—a viral attack—the judge and jury will determine whether or not it is guilty. Then, if another virus with the same RNA commits the same crime, the judge has precedent—in the form of CRISPR spacer sequences—to convict the new virus more efficiently.

In 2012, two groups of scientists realized that the bacterial mechanism of targeting and cutting DNA could be used in other species, including humans, by manually programming the crRNA to target any desired DNA sequence. Making the process even simpler, Nobel Prize winners Emmanuelle Charpentier, Jennifer Doudna, and their colleagues found a way to combine the crRNA and tracrRNA into a single guide RNA (sgRNA) (Gasiunas et al. 2012; Jinek et al. 2012). The discovery allowed three groups of scientists in 2013 to figure out methods to conduct CRISPR/Cas9 gene editing in-vivo, or in the living organism (Cong et al. 2013; Jinek et al. 2013; Mali et al. 2013; Seladi-Schulman 2019). Previously, all CRISPR/Cas9 experiments had been conducted in-vitro, outside living organisms, in test tubes or Petri dishes (Seladi-Schulman 2019).

An Illustration Of Genetic Engineering With CRISPR Cas9

Source: ARK Investment Management LLC, 2023. Illustration by Andrew Kim.

What Happens During Gene Editing?

Importantly, the actual gene-editing process is much the same for all gene-editing methods, as shown below.

An Illustration Of Gene-Editing

Source: ARK Investment Management LLC, 2023. Illustration by Andrew Kim.

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Today, CRISPR dominates gene editing in scientific literature and clinical trials. Compared ZFN and TALENs, CRISPR is more efficient, efficacious, and cost-effective.

Base Editing and Prime Editing

Because the risk associated with CRISPR is off-target edits, David Liu and post-doctoral researchers Alexis Komor and Nicole Gaudelli (Komor et al. 2016) built upon CRISPR/Cas9 and pioneered base editing in 2016. Altering only one base in a DNA sequence, scientists used base editing to attach another special enzyme to the Cas9 protein, targeting a specific base in a sequence and then switching to another base. As a result, the scientist did not introduce foreign DNA or rely on homology-detected repair (HDR, see footnote 5).

An Illustration Of Base Editing

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In 2019, David Liu and Andrew Anzalone further enhanced CRISPR/Cas9’s functionality (Anzalone et al. 2019). While base editing substitutes a single DNA base with each pass, prime editing allows for the insertion, deletion, and replacement of multiple bases in one pass. Rather than limiting the RNA to its role as the identifier of a correct DNA sequence, prime editing enables RNA to synthesize extra DNA bases on a targeted strand. In other words, prime editing offers all the benefits of base editing—no reliance on HDR or flawed repair mechanisms—with more flexibility. Relative to traditional CRISPR/Cas9, it offers more specificity (Anzalone et al. 2019).

An Illustration Of Prime Editing

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Base and prime editing have clear advantages over other gene-editing methods. Importantly, they do not cut chromosomes into pieces and cause double strand DNA breaks. Moreover, while traditional CRISPR Cas9 can disrupt genes, unlike prime and base editing it cannot correct them.

In the search for genetic disease cures, CRISPR/Cas9, base editing, and prime editing have taken center stage. Companies like CRISPR Therapeutics, Caribou Biosciences, Beam Therapeutics, and Prime Medicine are conducting trials to discern how these technologies might eliminate genetic disorders and improve upon human health. Researchers at the University of Michigan recently published their studies on the use of CRISPR/Cas9 to target brown fat cells in mice in hopes of targeting obesity in humans (Romanelli et al. 2021). Researchers at Harvard University and the Massachusetts Institute of Technology just developed a method of prime editing called TwinPE that allows scientists to insert or swap out entire genes in humans (Anzalone et al. 2021). Their lab also developed the first way to base-edit mitochondrial DNA (Mok et al. 2020). This year, the BBC reported that the Great Ormond Street Hospital (GOSH) in the United Kingdom demonstrated the potential of base editing by treating Alyssa, who had T-cell acute lymphoblastic leukemia and went into remission after being treated with the trial’s base-edited CARTs.[6]

Conclusion

While scientific progress over the past forty years has made gene editing more accurate, efficient, and cost-effective, many questions—especially about gene expression and traits—remain unanswered. Innovations in and around CRISPR/Cas9, base editing, and prime editing have improved gene editing, leaving scientists with many exciting opportunities to map the complex interrelationships in the human genome.

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