Cancer Letters

Cancer Letters

Volume 431, 1 September 2018, Pages 171-181
Cancer Letters

Original Articles
A CRISPR-Cas13a system for efficient and specific therapeutic targeting of mutant KRAS for pancreatic cancer treatment

https://doi.org/10.1016/j.canlet.2018.05.042Get rights and content

Highlights

  • An engineered CRISPR-Cas13a system can specially knock down mutant KRAS mRNA.

  • The Lw.Cas13a system can induce up to a 94% knockdown efficiency of KRAS-G12D mRNA.

  • Introducing a mismatch enabled the Lw.Cas13a system to specifically recognize KRAS-G12D.

  • Cas13a-mediated KRAS-G12D mRNA knockdown induced a dramatic cancer cell apoptosis.

  • Lw.Cas13a system can be used for the targeted therapy of other oncogenic mutations.

Abstract

Mutant KRAS is a known driver oncogene in pancreatic cancer. However, this protein remains an “undruggable” therapeutic target. Inhibiting mutated KRAS expression at the mRNA level is a potentially effective strategy. Recently, a novel CRISPR-Cas effector, Cas13a has been reported to specifically knock down mRNA expression under the guidance of a single CRISPR-RNA in mammalian cells. Here we demonstrate that the CRISPR-Cas13a system can be engineered for targeted therapy of mutant KRAS in pancreatic cancer. In initial screening, we show that the bacterial Cas13a protein and crRNA significantly knock down mutant KRAS mRNA expression, identifying a CRISPR-Cas13a system that can induce up to a 94% knockdown efficiency. Introducing a single mismatch into the crRNA-target duplex enabled the CRISPR-Cas13a system to specifically recognize KRAS-G12D mRNA with no detectable effects on wild-type KRAS mRNA. More importantly, CRISPR-Cas13a-mediated KRAS-G12D mRNA knockdown potently induced apoptosis in vitro and elicited marked tumor shrinkage in mice. Our work describes an optimization strategy for the development of a CRISPR-Cas13a system to affect efficient and specific knockdown of the oncogenic mRNA, establishing the CRISPR-Cas13a system as a flexible, targeted therapeutic tool.

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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