We determined that the practical range of the expression vector length is in between 450 and 950 bp, where it reaches up to 62% efficiency of converting input linear double-stranded DNA (dsDNA) into the circular expression vectors the method could be also used with lower efficiency for somewhat longer fragments. Moreover, our method takes advantage of this difference in properties by using exonuclease to digest unreacted or misreacted linear DNA fragments, thereby purifying the circular DNA. ![]() Linear DNA lacks such stability, which is a significant barrier to utilizing it for gene delivery. Circularized DNA is resistant to exonuclease degradation in the cytoplasm 14. We have developed an efficient fully synthetic method and protocol that enables the production of circularized DNA containing expression elements ready for transfection in as little as 3 h, thereby eliminating the bacterial cloning steps. However, the preparation of plasmids is labor intensive and time consuming process that typically takes 2 days 2 (reference typical bacterial cloning steps in Supplementary Note 1). The approach (I), gene editing using plasmid transfection for transient expression is commonly used due to its conceptual simplicity, ease of handling, and the stability of plasmids. The choice of delivery method will depend on the specific requirements and the researcher’s preferences, which can include electroporation 9, 10, lipofection 11, direct physical transfection 12, vector delivery on polymer particles and microcarriers 13, and modalities of the aforementioned methods. A variety of delivery methods from the aforementioned gene editing constructs are currently being used. ![]() The three most customary approaches that facilitate gene editing when delivered into cells are: (I) single or multiple plasmids 2 encoding Cas9 and other supplemental proteins and implementations of guide RNA suitable for traditional CRISPR/Cas9, base editing, prime editing or other methods 3, 4, 5, 6 (II) Cas9 mRNA and guide RNA 7 (III) Cas9 protein complexed with guide RNAs 8. Although we will discuss the specific implementation for gene editing and compare our method with some existing preparation techniques, the goal of this paper is to present a new approach to preparing the expression vectors in a laboratory environment-and not a more efficient gene editing method as such. However, since the principle behind this method is generic, this method can be useful for a variety of purposes and applications that require the expression of RNAs and proteins. As a demonstration, we will describe the use of our method in the gene editing application context. In this study, we present a new and efficient preparation method. Many techniques exist for the preparation and delivery of RNAs and proteins for transient expression in cell cultures 1. The method’s speed of preparation, low cost, and ease of use will make it a useful tool in applications requiring the expression of short RNAs and proteins. As proof of the principle, we applied Circular Vector expressing engineered prime editing guide RNA (epegRNA) in cell culture, and demonstrated matching and even exceeding performance of this method as compared to guides expressed by plasmids. The protocol describes how to take a linear double-stranded DNA fragment and efficiently circularize and purify this DNA fragment with minimal hands-on time. We have developed an efficient fully synthetic method and protocol that enables the production of circularized DNA containing expression elements ready for transfection in as little as 3 hours, thereby eliminating the bacterial cloning steps. ![]() While the expression plasmids can be designed and ordered from the contract manufacturers, the cost may be prohibitive when a large number of plasmids is required. The preparation of plasmids typically involves a laborious process of bacterial cloning, validation, and purification. DNA plasmids are an essential tool for delivery and expression of RNAs and proteins in cell culture experiments.
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