scott.pritchett

The CRISPR–Cas system has become one of the most widely used editing technologies for site-specific DNA manipulation owing to its simplicity and versatility. CRISPR-based tools typically consist of a nuclease that induces DNA double-strand breaks (DSBs) at a particular genomic sequence targeted by a guide RNA (gRNA). The DSBs are mostly resolved through one of the two major DSB repair pathways: non-homologous end joining (NHEJ) and homology-directed repair (HDR).

While versatile and easy to use, the traditional CRISPR Cas system suffers from off-target effects and size limitations.

Recently, novel and improved methods for gene editing such as base editing and prime editing have been introduced.

So how do these methods stack up to each other?

Gene Editing Methods

Figure 1(1). (A) Cas9 nuclease, guided by a single guide RNA (sgRNA), creates a targeted DNA double-strand break (DSB). With a repair template carrying desired edits (orange), the cellular homology-directed repair (HDR) machinery repairs the DSB, incorporating the template edits into the genome. (B) Base editors are engineered fusion proteins of nCas9 (D10A) and deaminase domains. Cytosine base editor (CBE) converts C to U in a single strand, and the resulting U:G heteroduplex can convert to T:A after DNA replication or repair. The uracil glycosylase inhibitor domain prevents U from reverting to C, favoring C to T conversion. Adenine base editor (ABE) deaminates A to form inosine (I), which pairs like G. The I:T heteroduplex can convert to G:C following DNA replication and repair. For both ABE and CBE, nCas9 (D10A) nicks the target strand to create an SSB; the mismatch repair (MMR) machinery pairs the deaminase-converted base correctly. Thus, CBE and ABE can install all four transition mutations (C to T, T to C, A to G, and G to A). (C) Prime editor is an engineered fusion protein of nickase Cas9 (nCas9) and a reverse transcriptase (RT). The nCas9 creates an SSB on the non-target strand, and the 3′ end hybridizes to the prime-editing guide RNA (pegRNA) and is reverse transcribed by the RT, incorporating the pegRNA-encoded edits (orange) into the new DNA strand. Equilibration between edited and unedited flaps, endogenous 5′ flap cleavage, ligation, and DNA repair result in stable genome incorporation of the desired edit.

• Reduced off-target effects: Less likely to cause unintended mutations
• Efficiency: High editing rates for certain types of point mutations

CRISPR-Cas9

CRISPR-Cas9 (Figure 1 A) is the original and most widely used CRISPR gene editing technique. It uses a gRNA to direct the Cas9 endonuclease to a specific DNA sequence, where it creates a DSB.

Advantages

• Versatility: Can be used for gene knockout, insertion, or replacement
• Efficiency: High editing rates in many cell types and organisms
• Multiplexing: Can target multiple genes simultaneously

Disadvantages

• Off-target effects: Can cause unintended edits at similar sequences
• Limited precision: Reliance on cellular repair mechanisms can lead to unpredictable outcomes
• Size limitations: Large Cas9 protein can be challenging to deliver into the cells

Applications of CRISPR-Cas9

Since its discovery twelve years ago, the CRISPR-Cas system has been used for many biotechnological and therapeutic applications. Many clinical trials are currently investigating if the mechanism can be used to induce mutations that can be of therapeutic value. For example, clinical trials are testing:

• The use of CRISPR-Cas9-Engineered T Cells in individuals who have relapsed or refractory multiple myeloma (ClinicalTrials.gov ID NCT04244656).
• The use of CRISPR-Cas9-Engineered T Cells in individuals who have advanced, relapsed or refractory renal cell carcinoma with clear cell differentiation (ClinicalTrials.gov ID NCT04438083)
• The safety and feasibility of using CRISPR/Cas9 in HIV patients to ablate the CCR5 gene and potentially avoid the development of AIDS (ClinicalTrials.gov ID NCT03164135).

Base Editing

Base editing (Figure 1 B) is a more precise gene editing method that doesn't rely on creating DSBs. It uses a catalytically impaired Cas9 fused to a deaminase enzyme. This complex can convert one DNA base to another (e.g., C to T or A to G) without cutting the DNA backbone.

Advantages

• Precision: Can make specific single-nucleotide changes without DSBs
• Reduced off-target effects: Less likely to cause unintended mutations
• Efficiency: High editing rates for certain types of point mutations

Disadvantages

• Limited scope: Can only perform certain types of base conversions
• Bystander edits: May edit other bases within the editing window
• PAM constraints: Requires specific protospacer adjacent motif (PAM) sequences near the target site

Applications of Base Editing for human disease management

• Base editing is being used by Verve therapeutics in a clinical trial for to treat heterozygous familial hypercholesterolemia (HeFH). HeFH is a genetic disorder associated with elevated levels of low-density lipoprotein cholesterol (LDL-C). The disorder results in an increased risk of developing heart disease⁵.
• Base editing can be used to potentially treat spinal muscular atrophy (Alves et. al.)⁶.
• Base editing can be used to potentially prevent infection or reinfection with the HIV virus (Knipping et al.)⁷.

Prime Editing

Prime editing (Figure 1 C) is the most recent and versatile gene editing method. By utilizing a prime editor protein (Cas9 nickase fused to a modified reverse transcriptase) and a prime editing guide RNA (pegRNA), this complex can directly “write” new genetic information into a specific DNA site.

Advantages

• Versatility: Can perform all types of small edits (insertions, deletions, and all base-to-base conversions)
• Precision: Highly specific with minimal off-target effects
• Flexibility: Less constrained by PAM requirements than other methods

Disadvantages

• Complexity: Requires careful design of pegRNAs
• Efficiency: Generally lower editing rates compared to Cas9 or base editors
• Size limitations: Large prime editor protein can be challenging to deliver into the cells; the system is not suitable for the insertion of large DNA fragments

Applications of prime editing in human disease management

• Prime editing is being used to investigate new modeling and treatment for genetic cardiac disease (Yao et. al.)⁸.
• Prime editing is being investigated for the treatment of inherited retinal diseases (Carvalho et. al.)⁹.
• Prime editing has the potential to generate the next generation of nanomedicines that address neurological disorders and neurodegenerative diseases, e.g. Parkinson’s disease, Alzheimer's disease, Amyotrophic Lateral Sclerosis, spinal muscular atrophy, etc. (BenDavid et. al.)¹⁰.

Applications of prime editing in staple crops

Areas of Future Discovery

CRISPR methodologies have revolutionized genetic engineering, offering unprecedented control over genetic material. While each gene editing method has its strengths and limitations, they collectively provide a powerful toolkit for researchers and potential therapeutic applications. As the field continues to advance, addressing some areas for improvement will likely lead to more precise, efficient, and versatile gene editing technologies, opening up new possibilities in basic research, biotechnology, and medicine.

Some of the areas of improvement in gene editing techniques include:

• Delivery methods: Developing more efficient and safer ways to deliver CRISPR components, especially for in vivo applications. This includes improving viral vectors and exploring non-viral delivery systems.
• Increasing efficiency: Enhancing the editing rates of prime editing and improving base editing for specific base conversions.
• Expanding targeting range: Developing Cas variants with more flexible PAM requirements to access more genomic sites.
• Reducing off-target effects: Further improving the specificity of all CRISPR methods, particularly for therapeutic applications.
• Overcoming size limitations: Creating smaller Cas proteins or split systems to facilitate delivery and packaging into viral vectors.
• Cell-type specificity: Developing methods to restrict editing activity to specific cell types or tissues.

The Future of Gene Editing

As research in the field progresses, gene editing techniques are likely to become more targeted and more effective increasing their use in fields such as disease management, gene therapy, and personalized medicine.

GenScript provides a complete line of services and products for CRISPR-Cas gene editing, from guide RNAs (sgRNA,crRNA,pegRNA to HDR templates and enzymes in protein , plasmid and RNA formats.

References

• [1]. Zhao, Z., Shang, P., Mohanraju, P., Geijsen, N. (2023). Trends in Biotechnology, 41:8.
• [2]. Kantor, A., McClements, M. E., & MacLaren, R. E. (2020). CRISPR-Cas9 DNA base-editing and prime-editing. International journal of molecular sciences, 21(17), 6240.
• [3]. Zhao, Z., Shang, P., Mohanraju, P., & Geijsen, N. (2023). Prime editing: advances and therapeutic applications. Trends in Biotechnology, 41(8), 1000-1012.
• [4]. Anzalone, A. V., Randolph, P. B., Davis, J. R., Sousa, A. A., Koblan, L. W., Levy, J. M., ... & Liu, D. R. (2019). Search-and-replace genome editing without double-strand breaks or donor DNA. Nature, 576(7785), 149-157.
• [5]. Verve therapeutics. https://ir.vervetx.com/news-releases/news-release-details/verve-therapeutics-announces-updates-its-pcsk9-program
• [6]. Alves,C., Ha, L., Yaworski, R., Lazzarotto, C., Christie, K., Reilly, A., Beauvais, A., Doll, R., Cruz, D., et al. Base editing as a genetic treatment for spinal muscular atrophy. bioRxiv 2023.01.20.524978.
• [7]. Knipping, F., Newby, G. A., Eide, C. R., McElroy, A. N., Nielsen, S., Smith, K., Fang, Y., Cornu, T., Costa, C., et al. (2022).Disruption of HIV-1 co-receptors CCR5 and CXCR4 in primary human T cells and hematopoietic stem and progenitor cells using base editing. Molecular Therapy 30(1):130-144.
• [8]. Yao, B., Lei, Z., Gonçalves, M., Sluijter J. (2024). Integrating Prime Editing and Cellular Reprogramming as Novel Strategies for Genetic Cardiac Disease Modeling and Treatment. Current Cardiology Reports. 26(11):1197-1208.
• [9]. Carvalho, C., Lemos, L., Antas, P., Seabra, M. C. (2023). Gene therapy for inherited retinal diseases: exploiting new tools in genome editing and nanotechnology. Frontiers in Ophthalmology 19:3:1270561.
• [10]. BenDavid, E., Ramezanian, S., Lu, Y., Rousseau J., Schroeder A., Lavertu M., Tremblay J. P. (2024) Emerging Perspectives on Prime Editor Delivery to the Brain. Pharmaceuticals11;17(6):763.
• [11]. Lin, Q., Zong, Y., Xue, C., Wang, S., Jin, S., Zhu, Z., Wang, Y., Anzalone, A. V., Raguram, A., Doman, J. L., Liu, D. R., Gao, C. (2020). Prime genome editing in rice and wheat. Nat Biotechnology, 38(5):582-585.
• [12]. Jiang, Y. Y., Chai, Y. P., Lu, M. H., Han, X. L., Lin, Q., Zhang, Y., Zhang, Q., Zhou, Y., Wang, X. C., Gao, C., Chen, QJ. (2020). Prime editing efficiently generates W542L and S621I double mutations in two ALS genes in maize. Genome Biology 21(1):257.
• [13]. Lu, Y., Tian, Y., Shen, R., Yao, Q., Zhong, D., Zhang, X., Zhu, J. K. (2020). Precise genome modification in tomato using an improved prime editing system. Plant Biotechnology Journal 19(3):415-417.