Targeted gene editing technologies have opened up new frontiers for researchers to manipulate genomic sequences in almost all eukaryotic cells. As a result, scientists can easily develop animal models of pathological processes and isogenic cell lines which facilitate gene therapy and biomedical research.
One of the gene editing techniques that have revolutionised biomedical and genetic research is clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9.
However, as powerful as CRISPR is, biotechnologists are trying to develop next-gen techniques and unlock even greater potential for more precise and efficient gene editing.
What is Gene Editing?
Gene editing is the process of modifying the genetic makeup of a living organism. It selectively targets a genome sequence and modifies the traits by substituting, inserting, or deleting genetic materials.
This ability to exert control over genome sequence and manipulate the genetic code in a living cell has a range of applications – from developing genetically modified (GM) crops to treating hereditary disorders through gene therapy.
CRISPR-Cas9 Gene Editing Technique: A Brief Overview
CRISPR-Cas9 is a gene editing technique discovered in 2013. It stemmed from a naturally occurring antiviral system in certain bacteria and archaea against invading bacteriophages. CRISPR is the genetic sequence found in the genomes of the aforementioned prokaryotic cells that makes them immune to bacteriophages.
CRISPR-Cas9 includes two major elements: a guide RNA (gRNA) and Cas9 (CRISPR-associated protein 9).
The gRNA is made up of an RNA segment and attaches to a target sequence in a gene. To be more specific, the RNA segment in the gRNA consists of a motif called the protospacer that includes ~20 nucleotides.
The gRNA in this gene editing technique is customisable and can be designed to ensure the sequence of the ~20 nucleotide region complements the target gene sequence that needs to be edited.
It implies that this region serves as a guiding component for the Cas9 protein. On the other hand, Cas9 is an endonuclease enzyme that can cut a double-stranded DNA at a desired location, facilitating genome editing. The gRNA forms a gRNA-Cas9 complex.
The protospacer motif in the gRNA then binds to the target gene sequence in the genome to be edited. Thus, Cas9 can identify and bind to the site of interest in the targeted gene. The Cas9 serves as a molecular scissor and breaks the target DNA at the location guided by the gRNA.
It means that by enabling the gRNA to bind to the target gene sequence, the ~20 nucleotide region directs the Cas9 protein to the required location in the genome which is complementary to the gRNA. As a result, the gene editing process incites at the required site.
Once the double-stranded break (DSB) is induced by the Cas9 protein in the targeted DNA, either of the two DNA repair pathways – non-homologous end joining (NHEJ) and Homology-directed repair (HDR) gets triggered in mammalian cells.
HDR, in conjunction with CRISPR-Cas9, leverages a homologous region of the unedited DNA strand as a template. It enables accurate, targeted gene modifications, allowing researchers to introduce required repairs in the targeted genome or facilitating the correction of disease-causing mutations. By enabling researchers to exert more control over the gene modification process, HDR helps them with gene therapy.
On the other hand, if gene knockout, gene disruption, or functional genomic CRISPR screens are required, NHEJ is leveraged in tandem with CRISPR-Cas9. It is the most used and error-prone DNA repair process that can insert or delete base pairs, or create indels at the cut site without using a homologous DNA template to link the broken ends of a DSB DNA.
Benefits of CRISPR-9 Gene Editing Technique: Potentials in Clinical Use
Genetic Disease Correction
CRISPR-Cas9 genome editing technique has been proven to hold immense potential in treating inherited disorders caused by single gene mutations such as hemophilia, β-thalassemia, hemoglobinopathies, Duchenne’s muscular dystrophy (DMD), cystic fibrosis (CF), etc.
Even though it’s still in the preclinical trial phase, research is being conducted to implement CRISPR-Cas9 for clinical practices. For example, by employing CRISPR-Cas9, scientists could successfully repair the CFTR (cystic fibrosis transmembrane conductance regulator) gene in intestinal stem cell organoids derived from two CF patients.
Treating HIV
CRISPR-Cas9 has demonstrated potential in treating infectious diseases, including HIV. Currently, though antiretroviral therapy is used to manage HIV, an effective therapeutic is yet to be discovered to treat this fatal disease that causes the virus to get injected into the affected gene permanently.
According to a recent study, CRISPR-Cas9 could be used to effectively target and disrupt the HIV-1 genome in latently infected cells, subduing gene expression and replication. Besides eradicating the HIV-1 infection, this RNA-directed gene editing technique can also be used to immunise uninfected cells against the viral infection.
Ex Vivo Genome Editing to Treat Malignancy or Other Diseases
Scientists and genome engineering exhibit growing interest in using CRISPR-Cas9 technology for ex vivo cell engineering to treat diseases such as cancer. In this process, T-cells and stem/progenitor cells are taken from a patient’s body and cultured artificially.
For a T-cell, it’s edited, altered, and then infused again into the patient’s bloodstream to fight against the target disease. For stem cells, they are differentiated into a target cell type and implanted back into the patient’s body to supersede the damaged/infected cells. This process has been proven to be highly targeted and effective since gene modification is done right on the cells.
Limitations of CRISPR-Cas9 Gene Editing Technology
Even though the CRISPR-Cas9 genome editing technique has revolutionised the field of genome engineering, it comes with some limitations:
Off-Target Effects
The high frequency of off-target mutations (≥50%) is a leading limitation of the CRISPR-Cas9 technique, especially for clinical applications. It’s a thing of concern as the frequency of OTE is greater than the intended mutation.
Caused by RGEN (RNA-guided endonuclease)-induced mutations, OTEs with this gene editing technique occur when the Cas9 protein creates breakups due to functioning on untargeted genomic sites other than the required on-target site. The outcome can be serious – genomic instability and disruption in functionality in non-target genes.
DNA-Damage Toxicity
Another leading limitation of the CRISPR-Cas9 technique is the possibility of inducing DNA damage toxicity. CRISPR-Cas9 requires the Cas9 protein to break the DNA on both sides, which can sometimes activate the P53 effect.
P53, often called the guardian of the genome, identifies any damage caused to the DNA during the manipulation process and stops the process by causing programmable death to that cell to ensure the error-prone gene cannot expand.
As a result, the efficacy of the processes drops. The concern heightens if the P53 protein in an edited cell cannot function properly. Failing to detect a cell carrying damaged DNA can favor the survival and growth of that cell.
Innumogenic Toxicity
The immunogenic responses induced by the CRISPR-Cas9 gene therapy process raise concerns among genetic engineers and scientists.
This is because many people carry preexisting adaptive immune responses to the commonly used Cas9 orthologs such as SaCas9 (S. aureus) and SpCas9 (S. pyogenes). In addition, an immune response can be induced by AAV (adeno-associated virus), which is often used to deliver CRISPR-Cas9 components.
To overcome this issue, experts are exploring other AAV serotypes. The aim is to figure out other immune-orthogonal combinations for repeated AAV-CRISPR gene therapy.
Next-Generation Gene Editing Techniques
There have been significant advancements made in the gene editing sector. Some next-gen gene editing tools and techniques are:
Base Editing
Invented by Dr. David Liu’s team in 2016, base editing is an advanced gene editing technique where CRISPR components fused with other enzymes to directly install point mutations into cellular RNA or DNA without breaking the double-stranded DNA.
In this process, a catalytically impaired Cas protein is blended with a base-modification enzyme to introduce precise modifications to specific DNA sequences. Two classes of base editors – cytosine base editors (CBEs) and adenine base editors (ABEs) – have been developed so far. While the CBE converts a C•G base pair into a T•A base pair, the ABE base editor does the opposite.
The gRNA in the Cas9 component directs the base editor to the site of interest, enabling precise and targeted editing of a single base.
Compared to the CRISPR genome editing technique, base editing shows more potential in precisely correcting genetic disorders by limiting the possibility of unintended mutation within the genome.
Prime Editing
Another next-gen gene editing technique that emerged as a way to overcome the limitations of the CRISPR-Cas9 system is prime editing. Developed in the lab of David R. Liu and detailed in Anzalone et al., this gene editing technique can offer more versatile and precise genome editing without creating a double-stranded DNA break (DSB).
Prime editors can virtually substitute any base for another, delete any required DNA section, or insert new DNA sequences into the site of interest within the targeted genome. The programmable nickase makes a single-strand DNA break, also called a nick, at the site of interest. From this point, the modification starts taking place.
In this process, the nickase needs to be minimally combined with an extended guide RNA (pegRNA) to precisely identify the site of interest. The pegRNA also provides the polymerase enzyme with the RNA template to help introduce the required modifications in the existing DNA sequence.
Compared to the traditional CRISPR system, prime editors enable more precise modification and point mutation while also helping dodge off-target mutations.
Ethical Considerations With Gene Editing
Since gene editing deals with altering the genetic makeup of a living cell, some ethical questions surround the processes. For example, gene therapy conducted on germ cells (eggs or sperm), also called germline therapy, can have a long-term impact.
The changes could be passed on to the forthcoming generations and could even adversely affect the fetus, leading to undiscovered consequences. Germline gene therapy is a controversial and hot topic in the genetic engineering field which raises questions about how ethical gene therapy is.
That being said, all challenges and ethical issues with gene editing technologies should be addressed carefully. In addition, these therapeutics need to be made as affordable and accessible as possible.
- SAFE Notes in Biotech: When They Work and When They Don’t - June 8, 2026
- Thyroid Eye Disease and Sinus Problems: Understanding the Connection and Treatment Options - April 19, 2026
- Best Payment Hubs for Banks and Financial Institutions in 2026: Compliance, Innovation, and Real-Time Readiness - March 24, 2026