Original ArticlesA CRISPR-Cas13a system for efficient and specific therapeutic targeting of mutant KRAS for pancreatic cancer treatment
Introduction
Pancreatic ductal adenocarcinoma (PDAC) is the most lethal malignancy among human cancers. There is an urgent need for the development of effective new therapeutic strategies for this disease [1]. Somatic mutations play a central role in the initiation and progression of cancer [2]. In recent years, many targeted drugs against these oncogenic mutations have been developed and have achieved excellent therapeutic effects. These drugs include osimertinib, in the treatment of EGFR-mutant lung cancer, and trametinib, in the treatment of BRAF-mutant melanoma [3,4]. The most frequent and important oncogenic mutation in pancreatic cancer is the mutant GTPase KRAS, which drives the initiation and progression of PDAC [[5], [6], [7], [8], [9]]. Unfortunately, direct and specific blockage of the KRAS protein with small molecule inhibitors remains a challenge [[10], [11], [12]]. An alternative strategy is the inhibition of the mutant KRAS at the transcriptional level, such as using RNA interference technology [[13], [14], [15], [16]]. However, RNA interference often elicits numerous non-specific, off-target effects [17]; a novel tool that can specifically distinguish mutant KRAS mRNA from the wild-type (WT) version and efficiently silence it is essential for targeted therapy of mutant KRAS.
Microbial clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated adaptive immune systems (CRISPR-Cas), the most popular of which is the CRISPR-Cas9 system, have been widely used for gene editing [[18], [19], [20], [21]]. Recently, a novel Cas enzyme, Cas13a (previously referred to as C2c2), was described to be a CRISPR RNA (crRNA)-guided RNA-targeting CRISPR effector [[22], [23], [24], [25], [26], [27], [28]]. Under the guidance of a single crRNA, Cas13a can bind and cleave a target RNA carrying a complementary sequence. Through this mechanism, the CRISPR-Cas13a system can effectively knock down mRNA expression in mammalian cells with a comparable efficacy and improved specificity over RNA interference technology [29,30]. In spite of these advantages, there have not yet been any reports of utilization of the CRISPR-Cas13a system in the field of cancer treatment.
In the present study, we demonstrate that the CRISPR-Cas13a system can be engineered for the efficient and specific knockdown of mutant KRAS-G12D mRNA in pancreatic cancer models. First, by selection of a bacterial species' Cas13a protein and screening crRNA positions, the CRISPR-Cas13a system resulted in up to a 94% knockdown efficiency of the KRAS-G12D mRNA. Second, we optimized the specificity of the CRISPR-Cas13a system by adding a single mismatch into the crRNA-target duplex. The optimized CRISPR-Cas13a system silenced up to 70% of KRAS-G12D mRNA expression, and exhibited almost no influence on WT KRAS mRNA. Finally, the Cas13a-crRNA complex effectively blocked the KRAS-driven signal pathways in multiple pancreatic cancer models that harbor the KRAS-G12D mutation, leading to apoptosis and tumor growth inhibition in vitro and in vivo.
Section snippets
Cas13a protein expression and purification
The full-length Lsh.Cas13a gene (encoding residues 1–1389) was synthesized from Sangon Biotech and cloned into the pET-30b vector (Novagen), which contains a His6 tag at the C-terminus of Lsh.Cas13a. The Lsh.Cas13a protein was overexpressed in E. coli Rosetta (DE3) (Novagen) cells that were induced with 0.1 mM isopropyl-1-thio-β-d-galactopyranoside (IPTG) at OD600 = 0.6 at 16 °C for 14 h. Cells were collected and lysed by sonication in buffer containing 20 mM Tris-HCl, pH 7.5, 500 mM NaCl and
Robust CRISPR-Cas13a-mediated knockdown of KRAS-G12D mRNA expression in pancreatic cancer cells
To achieve the highest possible silencing of KRAS-G12D mRNA using the CRISPR-Cas13a system, we expressed and purified three orthologous Cas13a proteins from Leptotrichia shahii (Lsh.Cas13a), Leptotrichia buccalis (Lbu.Cas13a) and Leptotrichia wadei (Lw.Cas13a; Fig. S1). In addition, we synthesized a series of crRNAs using in vitro transcription (Tables S1–S3). The crRNA for the CRISPR-Cas13a system is composed of a guide fragment and a scaffold fragment (Fig. 1A). The guide fragments in the
Discussion
Oncogenic mutations, particularly point mutations play an essential role in the initiation and progression of many types of cancer. Although using targeted therapy to inhibit oncogenic mutations has yielded enormous success, some key mutated oncogenes protein products are still regarded as “undruggable”, such as mutated KRAS [11]. In our present study, we established the CRISPR-Cas13a system as a novel tool for inhibiting mutant KRAS at the transcriptional level. By screening for an optimal
Author contributions
1. Conception and design: Xiao Zhao, Liang Liu, Yanli Wang, Guangjun Nie.
2. Development of methodology as follows:
Expression and purification of Cas13a proteins: Liang Liu, Xueyan Li.
Plasmid experiments: Jiayan Lang, Keman Cheng.
RNA expression analysis: Xiao Zhao, Yongwei Wang.
crRNA design and synthesis, cell transfection, cell function analysis, in vivo experiments: Xiao Zhao.
3. Acquisition of data: Xiao Zhao.
4. Analysis and interpretation of data: Xiao Zhao.
5. Writing, review and/or revision
Conflicts of interest statement
No potential conflicts of interest were disclosed.
Acknowledgements
No potential conflicts of interest were disclosed. This work was supported by the National Basic Research Plan of China (2018YFA020035), the Key Research Program of the Chinese Academy of Sciences (KGZD-EW-T06), the Innovation Research Group of National Natural Science Foundation (11621505), the Chinese Postdoctoral Science Foundation (2017M610839), the Key Research Project of Frontier Science of the Chinese Academy of Sciences (QYZDJ-SSW-SLH022), the National Postdoctoral Program for
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