The CRISPR-Cas9 gene editing technology has become the pinnacle of innovation in synthetic biology, revolutionizing the field since its development in the early 2010s. It has been instrumental in advancing therapeutic research, agricultural biotechnology, and fundamental studies of gene function. It enables scientists to easily alter genomes while being cheaper and more versatile than previously present gene editing techniques. However, CRISPR-Cas9 does have its limitations. It can easily cut any specific pieces of DNA, but creating desired mutations within this DNA can be tricky and often toxic to the cells
Why Explore Alternatives to CRISPR-Cas9?
While CRISPR-Cas9 remains the dominant tool for genome editing, the search for alternatives stems from the need to address its limitations:
- Off-target effects: CRISPR-Cas9 occasionally cuts unintended parts of the genome.
- Delivery challenges: Its relatively large size complicates delivery to certain cells or tissues.
- Specificity limitations: Precise editing can be problematic in some scenarios.
- Cell toxicity: Introducing cuts into DNA can lead to cellular damage.
For these reasons, researchers are exploring alternative genome editing tools that may offer greater specificity, reduced size, or unique functionalities. These tools aim to address CRISPR-Cas9's limitations by reducing off-target effects, improving delivery to hard-to-reach tissues, and enabling more precise genome edits for therapeutic and industrial applications.
Here are eight alternatives to CRISPR Cas9 gene editing technology
Retron Library Recombineering (RLR) Platform
Other gene editing techniques such as retrons and recombineering are being used to introduce mutations easily without breaking the DNA. Scientists at the Wyss Institute for Biologically Inspired Engineering and Harvard Medical School have created a Retron Library Recombineering (RLR) platform that can create millions of mutations while tagging them so everything can be screened at once.
RLR is now being used to explore bacterial genome editing applications, particularly for studying antibiotic resistance and microbiome engineering. For example, a study by the Wyss Institute demonstrated how RLR was utilized to create targeted mutations in E. coli strains to combat antibiotic resistance genes. Additionally, microbiome-focused companies are leveraging RLR to engineer beneficial bacterial strains for gut health applications. This makes it a promising tool in precision medicine and synthetic biology research.
The CRISPR-Cas9 technology uses a single guide RNA news to create genomic cuts in unwanted sites. Hera BioLabs have created a high-precision Cas-CLOVER gene editing system with undetectable off-target activity, supported by studies showing off-target effects reduced to below 0.1% in controlled experimental conditions. This precision is achieved through advanced computational design and the utilization of dual guide RNAs. that can be used as an alternative to CRISPR/Cas9. It achieves this specificity through the utilization of two guide RNAs. Cas-CLOVER is gaining traction in agricultural biotechnology, being used to engineer crop plants with improved traits like drought resistance and higher yields, addressing global food security challenges.
Mini-Cas9 Enzymes
The Cas9 enzyme and the single RNA strand are too large to function in the viruses used for gene therapy. Researchers have responded by creating a mini-Cas9 from the bacterium Staphylococcus aureus, which is small enough to fit inside of these viruses and has been tested in mice to correct the gene responsible for muscular dystrophy. In 2025, mini-Cas9 systems are being optimized for use in human clinical trials. Applications include correcting genetic mutations associated with rare diseases such as Huntington’s disease and cystic fibrosis.
NgAgo Protein Gene Editing System
Researchers have also unveiled a new gene-editing system using the NgAgo protein to cut DNA at a specific site without requiring a guide RNA. This approach is being worked on and provides hope that proteins from the Argonaute family could provide a better alternative to crispr technology gene editing. While initial excitement around NgAgo waned due to reproducibility issues, renewed research in 2024 demonstrated potential applications in bacterial editing, particularly in species resistant to CRISPR technologies.
CRISPR-Cpf1 System
The CRISPR-Cpf1 serves as a viable alternative to CRISPR-Cas9 as it is much easier to deliver into the cells and tissues due to its smaller size than CRISPR-Cas9. This is because it requires only one RNA whereas CRISPR-Cas9 requires two. It also cuts the DNA with more precision, enabling researchers to integrate DNA more efficiently. Now referred to as CRISPR-Cas12a, this system has found new applications in diagnostics. In 2025, Cas12a is being deployed in point-of-care diagnostic kits for detecting diseases like COVID-19 and cancer biomarkers, leveraging its precise DNA recognition. It exemplifies how crispr technologies are becoming central to modern diagnostic innovations.
These were the first endonucleases that could recognize and cleave target DNA at specific positions. They are made up of around 30 amino acid modules that interact with nucleotide triplets and can recognize long DNA sequences providing on-target specificity. NovoHelix offers gene editing services using ZFNs on animal models. ZFNs are increasingly being used in therapeutic applications. In 2025, ZFNs are part of ongoing clinical trials targeting hemophilia and sickle cell anemia, with institutions like St. Jude Children's Research Hospital leading the efforts. These trials showcase their potential for curing inherited blood disorders. These types of gene editing tools remain critical in precision medicine.
Transcription activator-like effector nucleases (TALENs)
These are restriction enzymes engineered to cut specific DNA sequences made by fusing a TAL effector, which can be engineered to bind to any desired DNA sequences. When combined with a suitable nuclease, these TALENs can easily cut DNA at specific locations. CROs like Biocytogen perform TALEN design and validation services that include mouse model designing and genotypic identification. TALENs are now widely used in regenerative medicine, especially for engineering stem cells for therapeutic purposes. Biocytogen has expanded its services to include TALEN-based solutions for gene therapy research.
FANA Antisense Oligonucleotide (FANA ASO) technology
AUM Biotech's FANA Antisense Oligonucleotide (FANA ASO) technology can be used to silence or regulate mRNA, microRNA, etc. The FANA oligos do not need any delivery agents or transfection reagents and can work on both in vitro and in vivo models. The platform is easy to optimize, non-toxic, and cost-effective. FANA ASO technology is being applied in 2025 to target neurodegenerative diseases such as ALS and Alzheimer’s, offering new hope for conditions with limited treatment options. This approach stands out due to its ability to silence specific RNA molecules without the need for delivery agents, ensuring minimal toxicity. Unlike other therapies, FANA ASO can cross the blood-brain barrier effectively, making it particularly suited for addressing central nervous system disorders. This represents a growing field of gene editing examples within therapeutic research.
Lab Management Software for CRISPR Cas9 alternative technologies providers: Scispot
Scispot has become the primary lab management software of choice for numerous modern biotech companies, including CRISPR-Cas9 gene editing alternative technology providers. Scispot creates a connected digital replica of these innovative bioscience companies. It centralizes their company-wide data, templatizes routine research, and automates non-scientific tasks. It makes other lab software such as electronic lab notebooks (ELNs) and lab information management systems (LIMS) redundant.
These providers use Scispot's operating platform to manage their research projects, inventory, samples, and partners all in one spot. In 2025, Scispot has added advanced integrations for high-throughput sequencing instruments, AI-powered data analytics, and automation features tailored for gene editing workflows. It supports crispr open source research projects and aligns with advancements in the editas pipeline and crispr app developments.
These providers use Scispot's operating platform to manage their research projects, inventory, samples, and partners all in one spot. Request a demo to learn how you can accelerate your research using Scispot.
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