Method
A simple and efficient method for CRISPR/Cas9-induced mutant screening

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Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system provides a technological breakthrough in mutant generation. Several methods such as the polymerase chain reaction (PCR)/restriction enzyme (RE) assay, T7 endonuclease I (T7EI) assay, Surveyor nuclease assay, PAGE-based genotyping assay, and high-resolution melting (HRM) analysis-based assay have been developed for screening CRISPR/Cas9-induced mutants. However, these methods are time- and labour-intensive and may also be sequence-limited or require very expensive equipment. Here, we described a cost-effective and sensitive screening technique based on conventional PCR, annealing at critical temperature PCR (ACT-PCR), for identifying mutants. ACT-PCR requires only a single PCR step followed by agarose gel electrophoresis. We demonstrated that ACT-PCR accurately distinguished CRISPR/Cas9-induced mutants from wild type in both rice and zebrafish. Moreover, the method can be adapted for accurately determining mutation frequency in cultured cells. The simplicity of ACT-PCR makes it particularly suitable for rapid, large-scale screening of CRISPR/Cas9-induced mutants in both plants and animals.

Introduction

Genetic mutants are critical for studying gene functions and analysing epistatic associations in genetic pathways. The recent development of sequence-specific nuclease systems such as transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), and particularly the type II clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has greatly expanded our ability to generate mutants at defined loci in many organisms (Li et al., 2013, Shan et al., 2013, Sander and Joung, 2014). The CRISPR-Cas9 system employs the CRISPR-associated endonuclease Cas9 and a single-guide RNA (sgRNA) to generate double-strand breaks (DSBs) at the target DNA site, subsequently leading to genetic mutations because of non-homologous end-joining (NHEJ) repair. Random insertion or deletion (indel) mutations induced by the CRISPR/Cas9 system usually occur proximate to the DSB site at 3 bp upstream of the protospacer-adjacent motif (PAM) (Cong et al., 2013). With the rapid popularity of genome editing in biological research, investigations regarding mutant isolation have significantly increased, particularly for large-scale screening.

To screen mutants produced by the CRISPR/Cas9 system, several approaches have been undertaken, such as the polymerase chain reaction (PCR)/restriction enzyme (RE) assay, T7 endonuclease I (T7EI) assay, Surveyor nuclease assay, PAGE-based genotyping assay, and high-resolution melting (HRM) analysis-based assay (Montgomery et al., 2007, Cong et al., 2013, Shan et al., 2014, Thomas et al., 2014, Zhu et al., 2014). However, each of these approaches has limitations such as being time- and labour-intensive, being sequence-limited, or requiring very expensive equipment. The PCR/RE assay is generally simple and sensitive for screening CRISPR/Cas9-induced mutants but is constrained by the availability of restriction enzyme sites near the target sequences. In contrast, the T7EI and Surveyor nuclease assays are wildly used and suitable for any target sequence as both can recognize and digest mismatched heteroduplex DNA; however, their detection sensitivity is lower than that of the PCR/RE assay, with both methodologies being time- and labour-intensive (Cong et al., 2013, Shan et al., 2014). The PAGE-based genotyping assay, in which heteroduplex and homoduplex DNAs formed by brief denaturation and annealing of PCR-amplified genomic regions spanning the mutations are detected using native PAGE, remains time-consuming and is not high-throughput (Zhu et al., 2014). The HRM analysis-based approach based on the difference in melting temperature (Tm) between PCR amplicons with or without mutations can be successfully used for mutagenesis screening, but requires expensive equipment (Montgomery et al., 2007, Thomas et al., 2014). Several modified methods have also been developed for detecting CRISPR/Cas9-induced mutants that utilize microfluidic capillary electrophoresis or fluorescent PCR (Ramlee et al., 2015, Chenouard et al., 2016, Kc et al., 2016); however, these methods are expensive. A PCR-based method for detecting indel mutations in zebrafish has been recently reported (Yu et al., 2014), but it has limited sensitivity for detecting 1-bp indel mutations, which is the most common mutation type in CRISPR/Cas9-treated plants.

Here, we describe a simple, accurate, ultra-fast and inexpensive solution, annealing at critical temperature PCR (ACT-PCR), for detecting CRISPR/Cas9-induced mutations in both plants and animals. Moreover, by integration with real-time PCR, ACT-PCR can be used to accurately quantify mutation frequency in cultured cells.

Section snippets

Schematic overview of ACT-PCR

PCR is a widely used technique that is capable of screening a large number of samples in a short time with high specificity. A single PCR cycle comprises three steps: denaturation, annealing and extension. The annealing temperature is critical for primer-template pairing for the success of PCR. An optimal temperature suppresses mismatched annealing, thereby reducing the generation of non-specific products. On the basis of this theory, we developed ACT-PCR to detect CRISPR/Cas9-induced mutants

Discussion

In this study, we developed ACT-PCR to identify CRISPR/Cas9-induced mutants. In contrast to other methods for mutant identification, ACT-PCR requires only conventional PCR and agarose gel electrophoresis (Table 1). Moreover, ACT-PCR is highly sensitive for identifying CRISPR/Cas9-induced mutants, even 1-bp indels. Compared with the PCR/RE assay, ACT-PCR can be utilized for detection regardless of available restriction enzyme sites. Moreover, unlike HRM analysis, ACT-PCR does not require

Plant materials

The process of generating mutants by the CRISPR/Cas9 system (Cas9 and Cas9 VQR variant) has been described in previous studies (Wang et al., 2015, Hu et al., 2016). Genomic DNA was extracted from rice using the cetyltrimethyl ammonium bromide (CTAB) method.

Zebrafish maintenance and embryo production

The zebrafish WT AB strain was grown in a 14-h/10-h light/dark cycle at 28°C. Embryos were produced by pair mating and maintained in culture dishes. One-cell-stage embryos were used for Cas9/sgRNA microinjection.

Preparation of Cas9 RNA and sgRNAs and mutagenesis screening in zebrafish

Cas9 RNA and gene-specific

Acknowledgments

We thank Prof. Jixian Zhai (Southern University of Science and Technology, China) for critical reviewing the manuscript. This study was supported by the National Natural Science Foundation of China (Nos. 31271681 and 3140101312) and the Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences.

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These authors contributed equally to this work.

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