Reviewing CRISPR/Cas9 technology
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system has revolutionized genome editing due to its high efficiency, broad applicability across diverse cell types and organisms, and strong therapeutic potential in cell and gene therapy (CGT). The CRISPR system is an adaptive immune mechanism used by bacteria and archaea to defend against invading genetic elements such as bacteriophages, plasmids, and transposons. Short sequences derived from these invading elements are integrated into the CRISPR locus and subsequently transcribed into guide RNAs (gRNAs), which direct Cas9 to identify and cleave complementary DNA sequences upon subsequent invasion. Based on this natural mechanism, researchers developed genome-editing tools by engineering gRNAs and harnessing the Cas9 proteins to induce site-specific DNA cleavage. Cas9 generates targeted double-strand breaks that are repaired by endogenous cellular DNA repair pathways, primarily non-homologous end joining (NHEJ) or homology-directed repair (HDR). Compared with earlier genome-editing technologies such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), CRISPR/Cas9 target specificity is determined by a short RNA sequence rather than complex protein engineering, enabling rapid retargeting to virtually any genomic locus by simply altering the gRNA. Owing to its fast design, ease of use, and low technical barrier, the CRISPR/Cas9 system has been widely adopted in experimental research. It functions efficiently in bacteria, plants, animals, and human cells, greatly accelerating advances across biology, medicine, and biotechnology. In addition, the low cost of gRNA synthesis enables large-scale and high-throughput genome-wide screening. In recent years, the CRISPR platform has been further expanded into derivative technologies, including base editing, prime editing, CRISPR interference (CRISPRi), CRISPR activation (CRISPRa), and epigenome editing.
CRISPR Delivery Methods
The success of CRISPR-based genome editing heavily relies on efficient delivery systems, which impact transfection efficiency, target specificity, and therapeutic potential. CRISPR delivery systems involve two essential components, the cargo (Cas nuclease, guide RNA, DNA donor, and regulatory elements) and the delivery vehicle (the strategies to transport these cargos into cells).
There are three main formats of the cargoes in CRISPR Delivery Systems as follows.
Plasmid DNA - one or two plasmids encoding Cas nuclease and gRNA
mRNA+gRNA - Cas nuclease mRNA co-delivered with a separately synthesized gRNA
RNP - A pre-assembled ribonucleoprotein (RNP) complex, composed of Cas nuclease and gRNA
These CRISPR cargos are typically delivered using three methods, physical methods (electroporation and microinjection), viral vehicles (lentivirus and AAV), and non-viral vehicles (LNPs, nanoparticles, and micelleplexes). While the delivery methods for the three cargo formats are flexible, certain cargo-vehicle pairings are preferred. Plasmid-based CRISPR is frequently delivered using viral or non-viral vectors, mRNA is commonly delivered via lipid nanoparticles or electroporation, and RNPs are most often introduced by electroporation, particularly for ex vivo genome editing. Early CRISPR studies predominantly relied on plasmid-based delivery due to its simplicity and accessibility; however, the field has increasingly shifted toward RNP-based approaches, driven by their rapid editing kinetics, reduced off-target effects, lack of vector integration risk, and improved safety profiles.
What we recommended to use
For CRISPR cell products and services, please view our genome-edited cells and cell line development service.
References
[1] C. E. Joseph, A. Jain, M. O. Yaqub, and L. K. Edison, “CRISPR-Cas Systems: Bridging Bacterial Immunity and Host Interactions,” Appl. Microbiol., vol. 5, no. 4, p. 118, Oct. 2025, doi: 10.3390/applmicrobiol5040118.
[2] S. Abbas, A. Saeed, M. Bibi, S. Perveen, and N. Masood, “CRISPR-Cas: From bacterial immunity to precision genome engineering,” Gene Rep., vol. 40, p. 102296, Sep. 2025, doi: 10.1016/j.genrep.2025.102296.
[3] H. Liao, J. Wu, N. J. VanDusen, Y. Li, and Y. Zheng, “CRISPR-Cas9-mediated homology-directed repair for precise gene editing,” Mol. Ther. Nucleic Acids, vol. 35, no. 4, p. 102344, Dec. 2024, doi: 10.1016/j.omtn.2024.102344.
[4] C. Xue and E. C. Greene, “DNA Repair Pathway Choices in CRISPR-Cas9-Mediated Genome Editing,” Trends Genet., vol. 37, no. 7, pp. 639–656, Jul. 2021, doi: 10.1016/j.tig.2021.02.008.
[5] A. Seijas, D. Cora, M. Novo, W. Al-Soufi, L. Sánchez, and Á. J. Arana, “CRISPR/Cas9 Delivery Systems to Enhance Gene Editing Efficiency,” Int. J. Mol. Sci., vol. 26, no. 9, p. 4420, May 2025, doi: 10.3390/ijms26094420.
