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Prime editing (PE), also known as pilot editing, as a new type of gene editing tool, is capable of generating precise substitutions, deletions, or insertions of DNA without producing DNA double-strand breaks, and has been widely used in fields such as basic research, agricultural breeding, and gene therapy, and is expected to enter human clinical trials.
The PE system consists of three components, nCas9, reverse transcriptase, and pegRNA. pegRNA contains, in addition to the guide RNA (sgRNA), the reverse transcriptase template (RTT) and the primer-binding site (PBS), which is required to produce the desired sequence 3′flap sequence. However, the current PE system has a number of challenges; delivery difficulties due to the system being too large; longer linear pegRNAs are unstable and susceptible to nuclease degradation. It is also limited by the need for G/C-rich at the target site, and high off-targetness.
Therefore, how to further improve the accuracy of PE systems, eliminate sequence constraints on editing sites and reduce the difficulty of delivery are important technical challenges that need to be solved in the field of genome editing.
In a research paper titled "Prime editing using CRISPR-Cas12a and circular RNAs in human cells," researchers developed four circular RNA-mediated lead editors (CPEs) using the smaller-sized Cas12a: niCPE. nuCPE and snuCPE, nuCPE, sniCPE, and snuCPE. compared to the Cas9-based lead editing system, the CPE system preferentially recognizes G/C-rich genomic regions and has multiple editing capabilities. Among them, the nuCPE and snuCPE systems based on nuclease showed a high editing efficiency of 10.42%, whereas the niCPE and sniCPE systems based on cutinase reached 24.89% and 40.75% editing efficiencies in human cells, respectively. In addition, the study showed that the niCPE and sniCPE systems could edit up to four genes simultaneously.
All of the efficient prime editors developed to date have used the CRISPR-Cas9 protein, which is derived from the type II prokaryotic CRISPR-Cas adaptive immune system. Many other prokaryotic nucleases have been identified, such as IsrB, IscB, TnpB, and various type V CRISPR-Cas proteins (including Cas12a). Cas12a (1227aa) has multiple advantages over Cas9 (1368aa): Cas12a recognizes A/T-rich regions of the genome and is capable of editing loci that cannot be edited by Cas9, and, with its lower off-targetness and smaller size, Cas12a enables more efficient delivery and facilitates multi-gene editing. However, because Cas12a has the characteristic of cutting pegRNAs, conventional pegRNAs are not suitable for the development of Cas12a-based guide editors.
In this study, the team developed a guide editing system, CPE, mediated by circular RNA (circRNA) using Cas12a, including nuclease-dependent CPE (niCPE), nuclease-dependent CPE (nuCPE), split nuclease-dependent CPE (sniCPE ) and split nuclease-dependent CPE (snuCPE).
The experimental results showed that the editing efficiencies of niCPE and sniCPE were higher than those of nuCPE and snuCPE, but nuCPE and snuCPE also showed satisfactory editing effects in human cells. Specifically, the efficiencies of niCPE and nuCPE were as high as 24.89% and 10.42%, respectively, in the human cell line HEK293T, which is suitable for large molecular weight delivery systems such as lentivirus. sniCPE and snuCPE were as high as 40.75% and 3.19%, respectively, in HEK293T cells, which is suitable for adeno-associated virus (AAV) delivery systems. In addition to HEK293T cells, niCPE and sniCPE were also effective in generating precise guide editing in HeLa, N2A, MCF7 and other cells.
In addition, the research team tandemized CRISPR RNA (crRNA) targeting different gene loci in the expression frame of the circular RNA, and then tandemized RTT-PBS sequences also in the expression frame of the circular RNA for multiple editing with the CPE system. The results showed that niCPE and sniCPE could perform double, triple and quadruple gene editing, and the editing efficiencies of sniCPE at the four loci ranged from 1.09% to 15.08%. The off-target effect of the CPE system was further detected, and the results showed that CPE had excellent specificity and almost no off-target effect was detected.
Overall, this study developed Cas12a-based cyclic RNA-mediated guide editing (CPE) systems that offer a selectable targeting range, multi-gene editing capability, and smaller size, which are advantageous for packaging and delivery of CPE systems. CPE systems with low off-target effects and high editing efficiency provide a universal paradigm for the development of novel guide editing systems utilizing various nuclease enzymes, and multiple types of CPE systems will have great potential in multiple scenarios, such as biological research, disease therapy, and crop breeding.
Creative Biogene is proud to provide CRISPR-related products and services for gene editing to customers worldwide. In addition, we specialize in providing premade adeno-associated virus particles, lentiviral particles, AAV, and lentiviral packaging services. Our customized solutions facilitate our customers to make progress in their research fields.
View more of our premade AAV particles, premade adenovirus particles and premade lentivirus particles.
In the future, we will continue to innovate to develop highly efficient AAVs to help more research on gene editing tools.
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