Shanghai Jiaotong University Develops New Method for Screening, Typing and Editing Efficiency Evaluation of Gene Edited Crop Mutants (SMART and Cc-qPCR)

Recently, Analytical Chemistry and Sensors and Actuators: B.
Chemical published online the research results of Professor Yang Litao's group of Zhang Dabing and the School of Life Science and Technology of Shanghai Jiao Tong University "One Versatile Cas9-Integrated Single-Tube Duplex Quantitative" Real-Time PCR System for Rapid Analysis of CRISPR/Cas-Induced Mutants" and "In vitro Argonaute cleavage-mediated quantitative PCR facilitates versatile CRISPR/Cas-induced mutant analysis"
。 Li Xueqi, Wang Yijie and postdoctoral fellow Li Rong, master's students of the School of Life Science and Technology, Shanghai Jiao Tong University, are the first authors and co-first authors, and Professor Yang Litao of the School of Life Science and Technology is the corresponding author
.
Professor Yuan Zheng and Professor Zhang Daming from the School of Life Science and Technology participated in the collaborative research
.

The CRISPR/Cas9 gene editing system has become one of the
main technical means for gene function research and rapid breeding of animals and plants.
CRISPR/Cas9 edits of target sites generally have different types of edits, such as heterozygous, homozygous, or chimeric genotypes
.
Therefore, how to quickly and accurately screen the mutant material generated by gene editing is very important
.
The team used the nucleic acid sequence-guided nuclease CRISPR/Cas9 and the prokaryotic Thermus thermophilus Argonaute (TtAgo) to fuse the in vitro genomic nuclease cleavage reaction with quantitative PCR and digital PCR, and established a multifunctional precise quantitative analysis method for screening, typing and efficiency of gene-edited crop mutants, which was successfully applied to the screening and analysis
of gene-edited rice mutants.

Cc-qPCR:

Based on the specific sequence recognition and cleavage characteristics of Cas9/sgRNA complex, a method combining CRISPR/Cas9 in vitro cleavage and dual qPCR detection was designed, named Cc-qPCR
.
The main principle of this method is to shear the genomic DNA of the sample to be measured with Cas9/sgRNA in vitro, then use the sheared genomic DNA as a template, amplify it by qPCR, and finally screen, type and quantify the sample to be tested according to the amplification results (Figure 1).

Principle of Cc-qPCR technology

In order to verify the detection ability of Cc-qPCR in actual samples, 24 gene-edited rice samples (W1-W24) were analyzed, 21 edited samples were successfully screened, and 17 were homozygous genotypes and 4 were heterozygous genotypes, and the screening and typing results were completely consistent
with Sanger sequencing results.
The gene editing frequency of the three mixed samples was quantified at 35.
82%, 8.
68% and 92.
10%, respectively, which was consistent with the expected results, indicating that Cc-qPCR could accurately calculate the frequency of
gene editing.

SMART：

Based on the specific sequence recognition and shearing characteristics of TtAgo/gDNA complex, a method combining TtAgo in vitro shearing and qPCR/ddPCR detection was designed, named SMART
.
The main principle of this method is to cut the genomic DNA of the sample to be tested with TtAgo/gDNA in vitro, then use the sheared genomic DNA as a template to amplify it with qPCR/ddPCR, and finally screen, type and quantify the sample to be tested according to the amplification results (Figure 2).

。 Using RdDM3l (RNA-directed DNA methylation 3-like) gene-edited rice mutant as the material, the feasibility and applicability of the SMART method were systematically verified, the working conditions of the SMART method were optimized, and the specificity, sensitivity and accuracy
of the SMART method were systematically evaluated.
The SMART detection results of the RdDM3l gene-edited rice offspring strain are consistent with the actual situation, and its sensitivity reaches 0.
5% in the mixed sample detection, which can accurately assess the gene editing efficiency
.
The results show that SMART can identify single-base replacement mutants with higher resolution and accuracy
than existing mutant detection methods.

Schematic diagram of how SMART works

The established Cc-qPCR and SMART methods, sgRNA and gDNA design are simple, and realize the screening of unknown gene editing mutants, mutation genotyping and evaluation of gene editing frequency
.
In the actual sample detection, it also fully demonstrates the advantages of high resolution, high accuracy and simple experimental operation, which is suitable for the rapid and accurate screening
of mutants in the process of molecular breeding.
Cc-qPCR and SMART methods also have the potential to be further used in the detection of
clinical mutant genes and tumor markers SNVs.

This research was supported
by the National Major Special Project, the Agricultural Field Project of the Shanghai Municipal Science and Technology Commission, and the Shanghai Oriental Scholars Program.

Links to papers:

Cc-qPCR paper link: https://pubs.
acs.
org/doi/10.
1021/acs.
analchem.
2c01837

SMART paper link: