CRISPR/Cas9: discovery, mechanism, and applications of the most advanced genome editing system

The acronym CRISPR/Cas9 stands for “Clustered Regularly Interspaced Short Palindromic Repeats / CRISPR-associated protein 9.” Initially identified as an adaptive defense system in many prokaryotes, it is now exploited as a high-precision genetic engineering tool. This technology enables targeted modification of cellular DNA by removing, adding, or replacing specific nucleotide sequences.

Although genome-editing methodologies were already available prior to the widespread adoption of CRISPR/Cas9 (for example, zinc-finger nucleases and TALENs), those tools are less efficient, more complex to design, and characterized by lower precision. The introduction of CRISPR/Cas9 therefore represented a substantial advance, opening application avenues that, until a few decades ago, belonged more to science fiction than to experimental practice.

1. Origins of the CRISPR system

The CRISPR system was first identified in 1987 within the genome of Escherichia coli, where regularly spaced repeat sequences separated by spacers—i.e., tracts of nonrepetitive DNA—were observed. Subsequent studies showed that these spacer sequences derive from fragments of viral genomes previously encountered by the bacterium or its progenitors. They constitute a form of molecular memory that enables recognition of future infectious agents. These sequences are actively transcribed by the bacterium and give rise to what is termed the guide RNA (gRNA).

The Cas9 protein, by contrast, is an endonuclease which—guided by the gRNA—introduces double-strand breaks in the target DNA, provided the latter contains a sequence complementary to the guide RNA. In the event of a recurrent infection, the gRNA pairs with the viral DNA and directs Cas9 to the precise cleavage site, leading to degradation of the viral genome and inactivation of the pathogen.

Understanding this mechanism led Emmanuelle Charpentier and Jennifer Doudna, in the early 2010s, to conceive the possibility of adapting CRISPR/Cas9 as a universal tool for genome editing in eukaryotic cells. The revolutionary impact of their discovery was recognized in 2020 with the award of the Nobel Prize in Chemistry, underscoring the enormous scientific and practical significance of this technology.

2. Mechanism of action of the CRISPR/Cas9 system

CRISPR/Cas9 activity relies on the specific recognition of the target DNA by a guide RNA (gRNA or sgRNA) designed to be complementary to a given genetic sequence, termed the protospacer. For Cas9 to bind and cut DNA, the presence of a short nucleotide motif adjacent to the protospacer, known as the PAM (Protospacer Adjacent Motif), is indispensable. In the case of Streptococcus pyogenes Cas9, the canonical PAM is the sequence 5′-NGG-3′. Cas9 induces a double-strand break (DSB) that typically occurs three nucleotides upstream of the PAM.

Once the DSB is introduced, the cell activates endogenous DNA repair systems that determine the final outcome of gene editing. There are two main pathways:

• Non-Homologous End Joining (NHEJ)
  Non-homologous end joining is a predominant mechanism in eukaryotic cells, characterized by speed but low fidelity. In the absence of a template strand, DNA ends are simply realigned and ligated, often with the insertion or deletion (indel) of a few nucleotides. Such errors can cause a reading-frame shift (frameshift), resulting in a truncated or nonfunctional protein. In molecular biology, NHEJ is intentionally exploited to silence genes or generate gene knockouts.

• Homology-Directed Repair (HDR)
  If a donor template—a homologous DNA sequence containing the desired correction or insertion—is provided, the DSB can be repaired via homologous recombination. This mechanism enables precise edits such as the correction of point mutations, insertion of reporter genes, or replacement of pathogenic alleles with functional versions. However, HDR is less efficient than NHEJ and is more active in specific cell-cycle phases (mainly S and G2, when chromatin is more accessible and recombination mechanisms are physiologically active).

Earlier genome-editing techniques, such as zinc-finger nucleases (ZFNs) and TALENs, required the design and synthesis of custom proteins for each target, with laborious, costly, and less flexible procedures. By contrast, the CRISPR/Cas9 system relies on a guide RNA, whose synthesis is simple, rapid, and economical. Moreover, the compact nature of the gRNA–Cas9 complex facilitates intracellular delivery via plasmids, viral vectors, or nonviral delivery systems, making it more versatile and scalable than previous methods.

3. Variants and optimizations of the CRISPR system

The CRISPR/Cas9 system derived from Streptococcus pyogenes (SpCas9) is the most widely used version for genome editing. However, in recent years numerous variants and adaptations have been developed to improve specificity, reduce off-target effects (unintended edits elsewhere in the genome), broaden applications, and enable more refined interventions on genomic sequence.

3.1. Cas9 protein variants

In addition to the wild-type form, several engineered versions of Cas9 exist:

• Cas9 nickase (nCas9): generated by mutating one of the two nuclease domains, it introduces a single-strand break (nick). If two nCas9 enzymes are directed to opposite, closely spaced positions, a DSB is obtained with greater specificity, as it is statistically less likely for two enzymes to make the same erroneous cut.

• Dead Cas9 (dCas9): inactivating mutations in both catalytic domains render the protein incapable of cutting DNA. However, dCas9 retains the ability to bind target sequences via the gRNA, enabling applications such as CRISPR interference (CRISPRi), in which gene expression is repressed, or CRISPR activation (CRISPRa), in which dCas9 is fused to transcriptional activators.

• High-fidelity Cas9 (eSpCas9, SpCas9-HF1, HypaCas9, and others): variants with mutations designed to reduce interactions with partially complementary sequences, thereby decreasing off-target effects without compromising efficiency.

Beyond Cas9, other endonucleases can be used within the CRISPR framework:

• Cas12a (Cpf1): recognizes a different PAM (typically 5′-TTTV-3′), introduces staggered cuts (cohesive ends), and employs a shorter gRNA, expanding targeting options.

• Cas13: unlike Cas9 and Cas12, it acts on RNA rather than DNA, making it useful for transcriptional knockdown and RNA-based diagnostic applications.

• Cas14: a smaller, versatile nuclease suitable for applications requiring reduced-size vectors.

3.2. Base editing

Base editing represents a significant evolution beyond simple DNA cutting. In this approach, Cas9 is used in its nickase form and fused to deaminase enzymes. This enables the precise conversion of one base into another (e.g., C→T or A→G) without introducing DSBs. Two types of deaminases are used: cytidine deaminases (CBEs), which convert cytosine (C) to uracil (U), and adenosine deaminases (ABEs), which convert adenine (A) to inosine (I), which is subsequently read as guanine (G) during replication.
Because base editing can change only a single nucleotide at a time, this technique is commonly used to create or correct single-nucleotide polymorphisms (SNPs) and point mutations, or to knock out genes.

3.3. Prime editing

Prime editing is a more recent technology (2019) that combines Cas9 nickase with a reverse transcriptase and an extended guide RNA (pegRNA). The nCas9 introduces a single-strand nick. The reverse transcriptase then synthesizes a new DNA segment using the pegRNA as template. The pegRNA is longer than conventional gRNAs because it contains both the reverse transcriptase–binding sequence and the DNA sequence to be inserted into the target. This system allows not only the correction of point mutations but also the introduction of insertions, deletions, or more complex substitutions without requiring DSBs.
In systems aimed at inserting a new sequence, what happens to the DNA strand that is not nicked? Immediately after the insertion of a new DNA sequence at the target site, the two DNA strands are not complementary in that region. However, during repair and subsequent replication, the cell resolves the mismatch because the introduced sequence is copied onto the complementary strand, thereby re-establishing base-pair complementarity.

4. Biomedical applications of CRISPR/Cas9

The CRISPR/Cas9 system has revolutionized biomedical prospects by enabling rapid, specific, and comparatively cost-effective manipulation of the genomes of cells and organisms. Applications span from generating experimental models to developing innovative therapies for genetic, infectious, and oncologic diseases.

CRISPR/Cas9 is particularly promising for monogenic disorders, which are caused by mutations in a single gene. Through NHEJ or HDR, it is possible to inactivate a pathogenic gene or correct the problematic mutation. Some examples include:

• Sickle cell disease and β-thalassemia: ex vivo clinical trials have demonstrated the system’s effectiveness in reactivating fetal hemoglobin (HbF), thereby compensating for defects in the β-globin chain.

• Leber congenital amaurosis (LCA10): early in vivo studies tested injection of CRISPR/Cas9 complexes to correct mutations in the CEP290 gene directly in retinal cells.

• Cystic fibrosis, Duchenne muscular dystrophy, and other rare disorders: preclinical research is underway to assess the feasibility of gene correction.

4.1. Significant clinical examples

Casgevy: the first approved CRISPR therapy
A historic milestone was reached in 2023 with approval—first by the U.S. Food and Drug Administration (FDA) and subsequently by European regulatory agencies—of the first therapy based on CRISPR/Cas9, named Casgevy (exagamglogene autotemcel, exa-cel).
Casgevy was developed for the treatment of sickle cell disease and transfusion-dependent β-thalassemia, both hereditary disorders caused by mutations in β-globin (HBB). Hemoglobin—responsible, among other functions, for transporting oxygen through the blood to tissues—is a protein composed of four smaller polypeptide chains: two β-globins and two α-globins. Mature forms of β-globin are produced after birth and replace fetal globin chains (γ-globin or HbF), which are produced only during fetal life. The γ-globin genes are silenced after birth when β-chain production begins. Researchers acted to disrupt the mechanism that represses fetal hemoglobin production. This strategy was considered simpler than repairing the defective β-globin gene, as mutations in that gene are numerous. With this approach, a mixture of fetal chains, β chains, and of course α chains is produced; the quantity of fetal chains generated is sufficient to restore overall hemoglobin function despite the continued presence of defective β chains.

The approach used is ex vivo:

1. Hematopoietic stem cells are collected from the patient.

2. In laboratory, CRISPR/Cas9 is used to inactivate a repressor (the BCL11A gene) that normally suppresses HbF expression.

3. Once reinfused, the modified cells produce sufficient HbF to compensate for the defect in adult hemoglobin.

Clinical studies have shown that, after treatment, most patients became free from vaso-occlusive crises (in sickle cell disease) or independent of transfusions (in β-thalassemia). This represents the first concrete demonstration of the therapeutic effectiveness of CRISPR in humans.

Personalized therapy for CPS1 deficiency
Another notable case was reported in 2025, involving the development of a personalized CRISPR/Cas9-based therapy for a newborn affected by a severe and rare metabolic disorder: carbamoyl-phosphate synthetase 1 (CPS1) deficiency.
This condition compromises the urea cycle, preventing the elimination of ammonia from the bloodstream and causing life-threatening hyperammonemia within the first days of life. Standard therapeutic options include a low-protein diet, the use of ammonia-scavenger drugs, and—in the most severe cases—liver transplantation; however, many patients do not survive long enough to undergo the procedure.

In this context, a team of researchers designed a CRISPR strategy termed k-ABE (“K” from the patient’s name—to underscore that this was a bespoke, single-patient approach—and “ABE” for adenine base editor), using lipid nanoparticles (LNPs) as the delivery vehicle to transport to the liver a Cas9 containing an adenine base editor together with the required gRNA. This Cas9 variant nicks only one strand of the target DNA to promote conversion of an A–T base pair to G–C, thereby restoring the correct sequence (the deficiency stems from a point mutation introducing a premature stop codon that yields a truncated, nonfunctional enzyme).

This is the first documented example of a personalized CRISPR therapy conceived and developed to measure for a single patient, attesting to the future potential of precision medicine based on genome editing.
Development proceeded through tests in hepatocyte cell lines carrying the patient’s mutation to screen different ABEs and gRNAs. Once the most effective combination was selected, the therapy was tested in mice and then in nonhuman primates, which also enabled dose calculation.
With a special FDA authorization, treatment was then administered to the child. After three administrations, the infant showed improved tolerance to dietary proteins and reduced need for ammonia-scavenger drugs.
The therapy was designed in just six months. Although it is still too early to speak of a definitive cure, the CRISPR/Cas9 system has begun to radically transform approaches to treating genetic diseases.

5. Applications in biotechnology and basic research

Beyond the clinical field, CRISPR/Cas9 is rapidly reshaping biological research and biotechnology:

• Development of experimental models: generating knockout or knock-in organisms (mice, zebrafish, Drosophila) has become much faster and more accessible than with previous techniques.

• Functional screening: genome-wide gRNA libraries enable systematic silencing of every gene in a genome, allowing identification of essential genes, disease-related pathways, or mechanisms of drug resistance.

• Agriculture: CRISPR is used to develop plant varieties resistant to pathogens (helping to substantially reduce pesticide use) or with improved nutritional traits.

• Industrial microbiology: engineering microorganisms that produce antibiotics, enzymes, or metabolites can optimize yields in industrial processes.

6. Limitations and challenges

Despite its enormous potential, the use of CRISPR/Cas9 does not come without limitations and critical issues. A primary concern is cutting specificity: Cas9 is not always perfectly faithful and can recognize and cleave sequences similar—but not identical—to the target, generating unintended mutations known as off-target effects. This phenomenon poses a major challenge, especially in clinical settings, where even minimal unintended changes elsewhere in the genome could have pathological consequences. To address this limitation, high-fidelity variants (such as SpCas9-HF1, eSpCas9, and HypaCas9) have been developed, and bioinformatic algorithms have been devised to more accurately predict potential off-target binding sites.

A second crucial limitation concerns the efficiency of HDR-mediated repair. Although this pathway is ideal for introducing precise modifications, the cell naturally favors NHEJ, which is simpler but less accurate. Moreover, HDR is primarily active during the S and G2 phases of the cell cycle, restricting the system’s potential for precise correction in tissues such as nervous or cardiac tissue, where most cells are post-mitotic.

Another obstacle relates to in vivo delivery. Introducing the CRISPR/Cas9 complex into target cells efficiently and safely remains one of the main challenges of gene therapy. Viral vectors—such as adeno-associated viruses (AAV) or lentiviruses—ensure high efficiency but carry risks of immunogenicity, undesired genomic integration, or cargo-size limitations. Conversely, alternative systems like lipid nanoparticles offer a better safety profile, sometimes at the expense of efficacy.

Immune responses also warrant consideration. Because Cas9 derives from common pathogenic bacteria such as Streptococcus pyogenes or Staphylococcus aureus, many individuals possess pre-existing antibodies or T cells capable of recognizing this protein as foreign. This could reduce treatment efficacy or, in the worst cases, trigger adverse reactions.

Finally, the potential of CRISPR inevitably raises ethical and regulatory questions. The use of genome editing in human germ cells and embryos is the subject of intense international debate: while it opens the possibility of preventing severe hereditary diseases, it also impacts ethical boundaries. It is therefore necessary to balance biotechnological progress with principles of ethical responsibility and transparency through appropriate legislation.

7. Future perspectives

The future of CRISPR/Cas9 looks extremely promising on multiple fronts. On the technical side, attention is focused on improving precision and versatility. Derivative technologies such as base editing and prime editing are already demonstrating the ability to introduce targeted modifications without generating double-strand breaks, thereby drastically reducing side effects and expanding the range of correctable mutations. In parallel, the ongoing discovery of new CRISPR nucleases in diverse microorganisms enriches the toolkit, providing smaller, more specific proteins better suited to different cellular contexts.

On the application front, personalized therapies are among the most exciting areas of development. The example of the base-editing treatment for a newborn with CPS1 deficiency has shown that CRISPR can be tailored to individual clinical needs, inaugurating an era of bespoke precision medicine. Looking ahead, one can envision a future in which patients with rare diseases gain access to therapies designed specifically to correct their unique mutations, without the need for lengthy, complex traditional drug discovery programs.

Another expanding frontier concerns molecular diagnostics. Certain variants, such as Cas12 and Cas13, possess collateral activities that can be harnessed as ultrasensitive biosensors for nucleic-acid detection. During the COVID-19 pandemic, these technologies were tested as rapid, low-cost diagnostic tools, opening the way for applications to other infectious diseases and environmental monitoring.

Ethical and regulatory considerations must not be overlooked. The pace of CRISPR’s development calls for clear, shared international guidelines to ensure safe and responsible use. The challenge will be to foster innovation while avoiding uncontrolled drifts that could undermine public trust or generate inequities in access to care.

In conclusion, although the CRISPR/Cas9 system is not without limitations, its transformative potential makes it one of the most powerful tools ever introduced into modern biology. In the coming years, it will likely be central not only to biomedical and biotechnological research but also to the cultural and ethical debate accompanying the genome-editing revolution.

REFERENCES AND FURTHER READING:

- CRISPR Genome Surgery in Stem Cells and Disease Tissues, (book), Edited by: Stephen H. Tsang, DOI: 10.1016/C2018-0-02310-5

- Laurent M, Geoffroy M, Pavani G, Guiraud S. CRISPR-Based Gene Therapies: From Preclinical to Clinical Treatments. Cells. 2024 May 8;13(10):800. doi: 10.3390/cells13100800. PMID: 38786024; PMCID: PMC11119143.

- Frangoul H, Altshuler D, Cappellini MD, Chen YS, Domm J, Eustace BK, Foell J, de la Fuente J, Grupp S, Handgretinger R, Ho TW, Kattamis A, Kernytsky A, Lekstrom-Himes J, Li AM, Locatelli F, Mapara MY, de Montalembert M, Rondelli D, Sharma A, Sheth S, Soni S, Steinberg MH, Wall D, Yen A, Corbacioglu S. CRISPR-Cas9 Gene Editing for Sickle Cell Disease and β-Thalassemia. N Engl J Med. 2021 Jan 21;384(3):252-260. doi: 10.1056/NEJMoa2031054. Epub 2020 Dec 5. PMID: 33283989.

- Musunuru K, Grandinette SA, Wang X, Hudson TR, Briseno K, Berry AM, Hacker JL, Hsu A, Silverstein RA, Hille LT, Ogul AN, Robinson-Garvin NA, Small JC, McCague S, Burke SM, Wright CM, Bick S, Indurthi V, Sharma S, Jepperson M, Vakulskas CA, Collingwood M, Keogh K, Jacobi A, Sturgeon M, Brommel C, Schmaljohn E, Kurgan G, Osborne T, Zhang H, Kinney K, Rettig G, Barbosa CJ, Semple SC, Tam YK, Lutz C, George LA, Kleinstiver BP, Liu DR, Ng K, Kassim SH, Giannikopoulos P, Alameh MG, Urnov FD, Ahrens-Nicklas RC. Patient-Specific In Vivo Gene Editing to Treat a Rare Genetic Disease. N Engl J Med. 2025 Jun 12;392(22):2235-2243. doi: 10.1056/NEJMoa2504747. Epub 2025 May 15. PMID: 40373211.

COVER IMAGE CREDIT: Foto di MJH SHIKDER su Unsplash