BRAF V600E mutation is a single-nucleotide variation (SNV) that is widely found in various cancers and has been demonstrated to have a strong association with the prognosis and development of some diseases.

Thus, we developed a strategy based on rolling circle amplification (RCA) and CRISPR/Cas14a to meet the great need for detecting highly specific BRAF V600E mutation in fine-needle biopsy samples. In this study, a padlock probe was designed to recognize and trigger subsequent ligase chain reactions (LCR).

And due to the Taq DNA ligase, a great number of ligated annular padlock probes were generated in the presence of BRAF V600E mutation, subsequently generating long repeated single-strand DNA by RCA.

The obtained amplicons were activators triggering the trans-cleavage of CRISPR/Cas14a. CRISPR/Cas14a shows outstanding performance in identifying ssDNA with single base mutation, which significantly increases the specificity of mutation discrimination. Under the optimal conditions, our strategy can identify BRAF V600E mutation down to 0.307 fM with a wide linear range from 1 fM to 10 pM. On the other hand, the dual identification strategy endows the method with terrific specificity for the detection of SNV. Furthermore, our method has been successfully employed to identify BRAF V600E mutation in clinical fine-needle aspiration samples, proving great potential for ultra-specific identification of low abundance BRAF V600E mutation and providing a novel method for diagnosis and treatment of cancer.

Cas14a strategy for BRAF V600E mutation.

fig1 Schematic illustration of the LCR-Cas14a strategy for BRAF V600E mutation.

The BRAF gene is a kinase-encoding gene that mainly regulates the MAP/ERK signaling pathway which affects cell growth, proliferation, and apoptosis. Single nucleotide mutation at position 1799 of the BRAF gene leads to a valine-to-glutamate substitution at residue 600 (V600E), resulting in constitutive activation of the RAF protein. Mutation of BRAF V600E has been identified in various cancers, including papillary thyroid carcinoma (PTC), non-Hodgkin lymphoma, colorectal cancer, melanoma, and non-small cell lung carcinoma. Some studies have demonstrated that BRAF V6000E is the most common and specific gene mutation in PTC and exhibited a strong association between both PTC recurrence and PTC-related mortality.

Fine needle aspiration (FNA) has been established as a universal method to diagnose thyroid nodules. However, cytologic assessment is not always accurate on account of non-diagnostic, indeterminate, and false-negative results. The analysis of BRAF V600E by fine needle biopsy (FNB) can improve the diagnostic accuracy of PTC compared with cytology alone.

Accordingly, precise and ultra-sensitive detection of BRAF V600E mutation will provide robust evidence for valuable diagnostic information and personalized therapy, which exerts a great benefit in predicting prognosis. Currently, various methods have been applied to identify BRAF V600E mutation, including the high-resolution melting (HRM) analysis, next-generation sequencing, Sanger sequencing, digital PCR, and immunohistochemistry. The gold standard method of molecular diagnosis for the BRAF V600E mutation is direct Sanger sequencing, which is specific and reliable. However, the sensitivity of Sanger sequencing was reported to identify only 7% to 20% of mutated alleles.

In addition, digital PCR and next-generation sequencing have improved the sensitivity to detect low abundance mutated DNA in clinical samples, but requirements for professionals and high-cost laboratory environments limit their widespread application. Thus, simple, highly specific, and ultra-sensitive methods for identifying BRAF V600E mutation are still desired.

The ligase chain reaction (LCR) has been used to detect single-nucleotide variation (SNV) due to its outstanding specificity in identifying single-base mismatch. During the LCR process, enzymatic ligation occurs only when the padlock probe is perfectly complementary to the target DNA via a hydrogen bond. Therefore, the LCR is capable of the identification of SNVs. However, LCR based methods mostly use synthetic DNA as the target, which cannot meet the increasing demand for the detection of real clinical samples with low levels of mutation copies within high abundance normal genomic DNA. The CRISPR/Cas system from prokaryotes called clustered regularly interspaced short palindromic repeats, which were widely applied for nucleic acid analysis. The CRISPR/Cas system can recognize a target nucleic acid through a 20 nt crRNA, which ensures precise nucleic acid targeting with minimal off-target binding. The crRNA can be programmed towards any interested DNA or RNA for hybridization with target nucleic acid to activate its multiple-turnover trans-cleavage activity to cleave nearby ssDNA reporters achieving sensitive and specific nucleic acid detection. Recently, Harrington et al. reported the Cas14a protein, a new type of a class 2 CRISPR effector that is about half the size of previously known class 2 CRISPR RNA-guided enzymes. Furthermore, compared with other CRISPR/Cas proteins such as Cas12a and Cas13a, Cas14a possesses several advantages for nucleic acid analysis and can recognize a single-stranded DNA (ssDNA) target in a PAM-independent manner and collaterally cleave random ssDNA for signal amplification. More importantly, Cas14a exhibits strong activity in the recognition of SNV when the mutation site appears in the middle of the ssDNA. Therefore, these excellent capabilities of Cas14a make it a potential tool for high-fidelity clinical detection of SNVs.

Herein, we establish a universal method combined with the LCR and CRISPR/Cas14a for highly specific detection of BRAF V600E mutation in clinical samples. After high-fidelity ligation of the padlock probe, long repeated single-strand DNA are generated by RCA, which is followed by the dual recognition of Cas14a for SNVs through its specific hybridization with amplicons. The dual identification by the LCR and Cas14a ensured the great specificity of our strategy. On the other hand, coupling the efficient amplification by RCA and the collaterally cleaving

activity of Cas14a provides a great signal augment that enables highly sensitive detection of low abundance BRAF V600E mutation. Moreover, we successfully applied the methods to the identification of BRAF V600E mutation in fine-needle aspiration samples, indicating that the proposed strategy is a promising tool for accurate molecular diagnosis.

Cas14a nuclease was obtained from Magigen Biotechnology Co. Ltd (Guangzhou, China).

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A dual identification strategy based on padlock ligation and CRISPR/Cas14a for highly specific detection of BRAF V600E mutation in clinical samples

Weicheng Shi, Yao Gong, Decai Zhang, Tiantian Yang, Ming Yi, Jingyi Tan,Shijia Ding and Wei Cheng