Dongguan Institute of Clinical Cancer Research, Affiliated Dongguan Hospital, Southern Medical University, Dongguan, 523058, China.
Cancer Research Institute, School of Basic Medical Sciences, State Key Laboratory of Organ Failure Research, National Clinical Research Center of Kidney Disease, Key Laboratory of Organ Failure Research (Ministry of Education), Southern Medical University, Guangzhou, 510515, China.
Genome Biol. 2023 Jun 23;24(1):145. doi: 10.1186/s13059-023-02992-z.
The CRISPR/Cas12a and CRISPR/Cas13d systems are widely used for fundamental research and hold great potential for future clinical applications. However, the short half-life of guide RNAs (gRNAs), particularly free gRNAs without Cas nuclease binding, limits their editing efficiency and durability.
Here, we engineer circular free gRNAs (cgRNAs) to increase their stability, and thus availability for Cas12a and Cas13d processing and loading, to boost editing. cgRNAs increases the efficiency of Cas12a-based transcription activators and genomic DNA cleavage by approximately 2.1- to 40.2-fold for single gene editing and 1.7- to 2.1-fold for multiplexed gene editing than their linear counterparts, without compromising specificity, across multiple sites and cell lines. Similarly, the RNA interference efficiency of Cas13d is increased by around 1.8-fold. In in vivo mouse liver, cgRNAs are more potent in activating gene expression and cleaving genomic DNA.
CgRNAs enable more efficient programmable DNA and RNA editing for Cas12a and Cas13d with broad applicability for fundamental research and gene therapy.
CRISPR/Cas12a 和 CRISPR/Cas13d 系统被广泛应用于基础研究，并在未来的临床应用中具有巨大的潜力。然而，向导 RNA（gRNA）的半衰期短，特别是没有 Cas 核酸酶结合的游离 gRNA，限制了它们的编辑效率和持久性。
在这里，我们设计了环状游离 gRNA（cgRNA）以提高其稳定性，从而增加 Cas12a 和 Cas13d 的加工和加载效率，从而提高编辑效率。cgRNA 可将 Cas12a 为基础的转录激活因子和基因组 DNA 切割的效率提高约 2.1 至 40.2 倍，用于单基因编辑，以及 1.7 至 2.1 倍，用于多重基因编辑，而不影响特异性，在多个位点和细胞系中。同样，Cas13d 的 RNA 干扰效率也提高了约 1.8 倍。在体内小鼠肝脏中，cgRNA 更有效地激活基因表达并切割基因组 DNA。
cgRNA 可实现更高效的可编程 DNA 和 RNA 编辑 Cas12a 和 Cas13d，具有广泛的基础研究和基因治疗适用性。
