Efficient Genome Editing Tool Research Articles

CRISPR-Cas system: a precise tool for plant genome editing

Biotic and abiotic stress affects the productivity and quality of the plant; it is necessarily important to develop improved crops that can withstand the environmental conditions. Conventional methods of breeding are very tedious process that require some alternative. The molecular approach of genetic modification emerged out as a precise method for plant breeding in the past few years. Genome is altered by removing, adding or substituting DNA bases in the host genome. Different methods of genome editing have been developed, including transcription activator like effector nucleases (TALENs), zinc-finger nucleases (ZFNs) and Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas. These nucleases have enabled researchers to manipulate gene of interest in crop plants. Among these CRISPR-Cas has been developed recently and found to be a more efficient tool for genome editing because of its simple design and low cost. In recent years, CRISPR-Cas9 has been utilized for gene knockouts and knock-ins, multiplex gene targeting, and gene regulation in plants. In this review, we provide the use of CRISPR–Cas9 technology in genome editing and its fruitful applications in plants specifically in terms of improvement in quality and resistance to biotic and abiotic stress. Designing of an efficient CRISPR–Cas system using bioinformatic tools have also been discussed.

Progression and application of CRISPR-Cas genomic editors.

Base editing technology is an efficient tool for genome editing, particularly in the correction of base mutations. Diverse base editing systems were developed according to the dCas9 or nCas9 linked with different deaminase or reverse transcriptase in the editors, including ABEs, CBEs, PEs and dual-functional of base editor (such as CGBE1, A&C-BEmax, ACBE, etc.). Currently, Base editing technology has been widely applied to various fields such as microorganisms, plants, animals and medicine for basic research and therapeutics. Here, we reviewed the advancement of base editing technology. We also discussed the application of base editors in different areas in the future.

CRISPR/Cas12a-mediated CHO genome engineering can be effectively integrated at multiple stages of the cell line generation process for bioproduction.

Most biopharmaceuticals produced today are generated using Chinese hamster ovary (CHO) cells, therefore significant attention is focused on methods to improve CHO cell productivity and product quality. The discovery of gene-editing tools, such as CRISPR/Cas9, offers new opportunities to improve CHO cell bioproduction through cell line engineering. Recently an additional CRISPR-associated protein, Cas12a (Cpf1), was shown to be effective for gene editing in eukaryotic cells, including CHO. In this study, we demonstrate the successful application of CRISPR/Cas12a for the generation of clonally derived CHO knockout (KO) cell lines with improved product quality attributes. While we found Cas12a efficiency to be highly dependent on the targeting RNA used, we were able to generate CHO KO cell lines using small screens of only 96-320 clonally derived cell lines. Additionally, we present a novel bulk culture analysis approach that can be used to quickly assess CRISPR RNA efficiency and determine ideal screen sizes for generating genetic KO cell lines. Most critically, we find that Cas12a can be directly integrated into the cell line generation process through cotransfection with no negative impact on titer or screen size. Overall, our results show CRISPR/Cas12a to be an efficient and effective CHO genome editing tool.

Base editing in rice: current progress, advances, limitations, and future perspectives.

Base editing is one of the promising genome editing tools for generating single-nucleotide changes in rice genome. Rice (Oryza sativa L.) is an important staple food crop, feeding half of the population around the globe. Developing new rice varieties with desirable agronomic traits is necessary for sustaining global food security. The use of genome editing technologies for developing rice varieties is pre-requisite in the present scenario. Among the genome editing technologies developed for rice crop improvement, base editing technology has emerged as an efficient and reliable tool for precise genome editing in rice plants. Base editing technology utilizes either adenosine or cytidine base editor for precise editing at the target region. A base editor (adenosine or cytidine) is a fusion of catalytically inactive CRISPR/Cas9 domain and adenosine or cytidine deaminase domain. In this review, authors have discussed the different adenine and cytosine base editors developed so far for precise genome editing of rice via base editing technology. We address the current progress, advances, limitations, as well as future perspectives of the base editing technology for rice crop improvement.

Implementing CRISPR-Cas12a for Efficient Genome Editing in Yarrowia lipolytica.

The oleaginous yeast Yarrowia lipolytica has emerged as an industrially relevant chassis to produce various valuable chemicals. Metabolic engineering of Y. lipolytica relies on the availability of geneticengineering tools. Existing engineering strategies for this yeast include homologous recombination, random integration, and episomal plasmid-based gene expression. CRISPR-Cas9 based genome-editing toolbox has also been developed to facilitate multiplexed gene disruption and regulation. Alternative to Cas9, the CRISPR effector Cas12a has also been adopted to perform genome engineering in multiple species. Due to its distinctive features such as short and simple crRNA structure, the ability to process its own crRNA and T-rich PAM sequence (TTTN), Cas12a holds promising potential to be developed as an efficient genome-editing tool. In this chapter, we describe the protocol to implement multiplexed genome editing in Y. lipolytica. The delivery of AsCas12a and crRNA expression via a single plasmid was described. CRISPR-Cas12a-based genome editing could expand the genetic toolbox of Y. lipolytica, whihc iscomplementary to the classical Cas9-based tools.

Genome-wide identification and analysis of highly specific CRISPR/Cas9 editing sites in pepper (Capsicum annuum L.).

The CRISPR/Cas9 system is an efficient genome editing tool that possesses the outstanding advantages of simplicity and high efficiency. Genome-wide identification and specificity analysis of editing sites is an effective approach for mitigating the risk of off-target effects of CRISPR/Cas9 and has been applied in several plant species but has not yet been reported in pepper. In present study, we first identified genome-wide CRISPR/Cas9 editing sites based on the ‘Zunla-1’ reference genome and then evaluated the specificity of CRISPR/Cas9 editing sites through whole-genome alignment. Results showed that a total of 603,202,314 CRISPR/Cas9 editing sites, including 229,909,837 (~38.11%) NGG-PAM sites and 373,292,477 (~61.89%) NAG-PAM sites, were detectable in the pepper genome, and the systematic characterization of their composition and distribution was performed. Furthermore, 29,623,855 highly specific NGG-PAM sites were identified through whole-genome alignment analysis. There were 26,699,38 (~90.13%) highly specific NGG-PAM sites located in intergenic regions, which was 9.13 times of the number in genic regions, but the average density in genic regions was higher than that in intergenic regions. More importantly, 34,251 (~96.93%) out of 35,336 annotated genes exhibited at least one highly specific NGG-PAM site in their exons, and 90.50% of the annotated genes exhibited at least 4 highly specific NGG- PAM sites, indicating that the set of highly specific CRISPR/Cas9 editing sites identified in this study was widely applicable and conducive to the minimization of the off-target effects of CRISPR/Cas9 in pepper.

In Vivo CRISPR/Cas9-Based Targeted Disruption and Knockin of a Long Noncoding RNA.

The CRISPR/Cas9 system has been widely used as an efficient genome-editing tool for studying physiological functions of long noncoding RNAs (lncRNAs). In this chapter, we describe the experimental procedures for using the CRISPR/Cas9 system to genetically modify a long noncoding RNA in vivo through the targeted disruption and knockin approaches.

Nobel Prize in Chemistry 2020: The great breakthrough from frontier research in microbiology

The 2020 Nobel Prize in Chemistry was awarded to Emmanuelle Charpentier and Jennifer A. Doudna for their joint development of the highly efficient CRISPR-Cas9 genome editing tools, which have taken the life sciences into a new era. The CRISPR-Cas system was first discovered in bacteria in 1987. After more than 20 years of arduous exploration of these enigmatic systems that were found in more than 90% archaea and about half bacteria, their antiviral function was finally confirmed in 2007. This has inspired more researchers to focus on the CRISPR-Cas systems, of which the general anti-virus mechanisms were also uncovered within the next few years, that the crRNA containing the virus sequence information guides the Cas protein(s) to recognize the corresponding virus and cleave their DNA or RNA. Among those pioneers in this field, Charpentier and Doudna mainly focused on the type II CRISPR-Cas9 system, which is much simpler than the wide-spread type I systems. In 2012, Doudna and Charpentier had elucidated the fine structure of the anti-virus effector complex of the CRISPR-Cas9 system, which contains a single Cas9 protein and two RNA components (crRNA and tracrRNA). It was this work that led to the breakthrough in developing the CRISPR-Cas9 genome editing tools. Significantly, they innovatively connected the tracrRNA and crRNA into a single RNA chimera (sgRNA), greatly simplifying the genome editing tools to only two components, the sgRNA and the single Cas9 protein. Compared with the genome editing tools developed in the past (Meganuclease, ZFN and TALEN), CRISPR-Cas9 has significant advantages such as easy programming, high efficiency and a wide application range. These merits make CRISPR-Cas9 so far the most widely used genome editing technique. In basic research, CRISPR-Cas9 genome editing has been used to elucidate the functions of unknown genes or pathways. In application, this technique has greatly improved the efficiency of animal and plant breeding, and made it possible to cure some genetic diseases and even cancers. Moreover, based on this CRISPR-Cas9 platform, other advanced genome editing tools have also been developed, such as base editing and prime editing, etc. It is also believed that, in addition to CRISPR-Cas systems, there are still many other undiscovered antiviral systems in microbes, as well as a large number of unknown nucleic acid recognition and processing elements, which are expected to optimize or upgrade the current genome editing tools, or even innovate entirely new genome editing tools. The CRISPR-Cas genome editing tools were developed from the bacterial anti-virus immune systems, thus the 2020 Nobel Prize in Chemistry is essentially a reward to the frontier research in microbiology. In this article, we will briefly introduce i) the timeline for the development of genome editing tools; ii) the historical background of the CRISPR-Cas research; iii) the breakthrough works for the birth of CRISPR-Cas9 genome editing technology. The future frontier research in microbiology and big data mining to further optimize or innovate genome editing tools is also prospected.

Nucleolus localization of SpyCas9 affects its stability and interferes with host protein translation in mammalian cells

The CRISPR/Cas9 system, originally derived from the prokaryotic adaptive immune system, has been developed as efficient genome editing tools. It enables precise gene manipulation on chromosomal DNA through the specific binding of programmable sgRNA to target DNA, and the Cas9 protein, which has endonuclease activity, will cut a double strand break at specific locus. However, Cas9 is a foreign protein in mammalian cells, and the potential risks associated with its introduction into mammalian cells are not fully understood. In this study, we performed pull-down and mass spectrometry (MS) analysis of Streptococcus pyogenes Cas9 (SpyCas9) interacting proteins in HEK293T cells and showed that the majority of Cas9-associated proteins identified by MS were localized in the nucleolus. Interestingly, we further discovered that the Cas9 protein contains a sequence encoding a nucleolus detention signal (NoDS). Compared with wild-type (WT) Cas9, NoDS-mutated variants of Cas9 (mCas9) are less stable, although their gene editing activity is minimally affected. Overexpression of WT Cas9, but not mCas9, causes general effects on transcription and protein translation in the host cell. Overall, identification of NoDS in Cas9 will improve the understanding of Cas9's biological function in vivo, and the removal of NoDS in Cas9 may enhance its safety for future clinical use.

Current breeding and genomic approaches to enhance the cane and sugar productivity under abiotic stress conditions.

Sugarcane (Saccharumspp.) crop is vulnerable to many abiotic stresses such as drought, salinity, waterlogging, cold and high temperature due to climate change. Over the past few decades new breeding and genomic approaches have been used to enhance the genotypic performance under abiotic stress conditions. In sugarcane, introgression of genes from wild species and allied genera for abiotic stress tolerance traits plays a significant role in the development of several stress-tolerant varieties. Moreover, the genomics and transcriptomics approaches have helped to elucidate the key genes/TFs and pathways involved in abiotic stress tolerance in sugarcane. Several novel miRNAs families /proteins or regulatory elements that are responsible for drought, salinity, and cold tolerance have been identified through high-throughput sequencing. The existing sugarcane monoploid genome sequence information opens new gateways and opportunities for researchers to improve the desired traits through efficient genome editing tools, such as the clustered regularly interspaced short palindromic repeat-Cas (CRISPR/Cas) system. TALEN mediated mutations in a highly conserved region of the caffeic acid O-methyltransferase (COMT) of sugarcane significantly reduces the lignin content in the cell wall which is amenable for biofuel production from lignocellulosic biomass. In this review, we focus on current breeding with genomic approaches and their substantial role in enhancing cane production under the abiotic stress conditions, which is expected to provide new insights to plant breeders and biotechnologists to modify their strategy in developing stress-tolerant sugarcane varieties, which can highlight the future demand of cane, bio-energy, and viability of sugar industries.

Design and Construction of Portable CRISPR-Cpf1-Mediated Genome Editing in Bacillus subtilis 168 Oriented Toward Multiple Utilities.

Bacillus subtilis is an important Gram-positive bacterium for industrial biotechnology, which has been widely used to produce diverse high-value added chemicals and industrially and pharmaceutically relevant proteins. Robust and versatile toolkits for genome editing in B. subtilis are highly demanding to design higher version chassis. Although the Streptococcus pyogenes (Sp) CRISPR-Cas9 has been extensively adapted for genome engineering of multiple bacteria, it has many defects, such as higher molecular weight which leads to higher carrier load, low deletion efficiency and complexity of sgRNA construction for multiplex genome editing. Here, we designed a CRISPR-Cpf1-based toolkit employing a type V Cas protein, Cpf1 from Francisella novicida. Using this platform, we precisely deleted single gene and gene cluster in B. subtilis with high editing efficiency, such as sacA, ganA, ligD & ligV, and bac operon. Especially, an extremely large gene cluster of 38 kb in B. subtilis genome was accurately deleted from the genome without introducing any unexpected mutations. Meanwhile, the synthetic platform was further upgraded to a version for multiplex genome editing, upon which two genes sacA and aprE were precisely and efficiently deleted using only one plasmid harboring two targeting sequences. In addition, we successfully inserted foreign genes into the genome of the chassis using the CRISPR-Cpf1 platform. Our work highlighted the availability of CRISPR-Cpf1 to gene manipulation in B. subtilis, including the flexible deletion of a single gene and multiple genes or a gene cluster, and gene knock-in. The designed genome-editing platform was easily and broadly applicable to other microorganisms. The novel platforms we constructed in this study provide a promising tool for efficient genome editing in diverse bacteria.

Efficient genome editing in pathogenic mycobacteria using Streptococcus thermophilus CRISPR1-Cas9

The ability to genetically engineer pathogenic mycobacteria has increased significantly over the last decades due to the generation of new molecular tools. Recently, the application of the Streptococcus pyogenes and the Streptococcus thermophilus CRISPR‐Cas9 systems in mycobacteria has enabled gene editing and efficient CRISPR interference‐mediated transcriptional regulation. Here, we converted CRISPR interference into an efficient genome editing tool for mycobacteria. We demonstrate that the Streptococcus thermophilus CRISPR1-Cas9 (Sth1Cas9) is functional in Mycobacterium marinum and Mycobacterium tuberculosis, enabling highly efficient and precise DNA breaks and indel formation, without any off-target effects. In addition, with dual sgRNAs this system can be used to generate two indels simultaneously or to create specific deletions. The ability to use the power of the CRISPR-Cas9-mediated gene editing toolbox in M. tuberculosis with a single step will accelerate research into this deadly pathogen.

A robust genome-editing method for wild plant species Nicotiana attenuata

CRISPR genome-editing techniques theoretically enable us to edit any genes in any plants. However, plant tissue culture is required for generating targeted mutants in plants, except in some model plant species such as Arabidopsis thaliana. To modify ecologically or agronomically important traits in plants using the CRISPR system, a robust plant regeneration method and efficient genome-editing tools must be developed. This study shows the entire process of genome editing and the regeneration process for wild tobacco, Nicotiana attenuata. We delivered T-DNA harboring Streptococcus pyogenes Cas9 (SpCas9) and guide RNA (gRNA) via Agrobacterium-mediated transformation into the hypocotyl cells of the wild tobacco and regenerated gene-edited plants. The efficacy of genome editing was measured in N. attenuata protoplasts in which SpCas9 and gRNA were transiently expressed. Light intensity (476.66–627.00 μW/cm2, 20.69–52.21 μE) was optimized to enhance the emergence of plant shoots during callus induction, and the core step of dealing with plant tissues was recorded. In addition, we found that wounding the bottom part of mature plants was critical for root regeneration. By tracking mutation patterns and efficiency at each regeneration step, we found that the mutation was induced early on in the tissue culture process and maintained throughout the regeneration process. Genome-editing techniques have opened the way to study the function of the genes in all plants, and this study will provide guidelines for editing a gene in the plant of interest.

High-throughput assessment of mutations generated by genome editing in induced pluripotent stem cells by high-resolution melting analysis

Background and aimsGenome editing of induced pluripotent stem cells (iPSCs) holds great potential for both disease modeling and regenerative medicine. Although clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 provides an efficient and precise genome editing tool, iPSCs are especially difficult to transfect, resulting in a small percentage of cells carrying the desired correction. A high-throughput method to identify edited clones is required to reduce the time and costs of such an approach. MethodsHere we assess high-resolution melting analysis (HRMA), a simple and efficient real-time polymerase chain reaction-based method, and compare it with more commonly used assays. Results and conclusionsOur data show that HRMA is a robust and highly sensitive method, allowing the cost-effective and time-saving screening of genome-edited iPSCs. Samples can be prepared directly from 96-well microtiter plates for high-throughput analysis, and amplicons can be further analyzed with downstream techniques for further confirmation, if needed.

Genome Editing for Rare Diseases.

Significant numbers of patients worldwide are affected by various rare diseases, but the effective treatment options to these individuals are limited. Rare diseases remain underfunded compared to more common diseases, leading to significant delays in research progress and ultimately, to finding an effective cure. Here, we review the use of genome-editing tools to understand the pathogenesis of rare diseases and develop additional therapeutic approaches with a high degree of precision. Several genome-editing approaches, including CRISPR/Cas9, TALEN and ZFN, have been used to generate animal models of rare diseases, understand the disease pathogenesis, correct pathogenic mutations in patient-derived somatic cells and iPSCs, and develop new therapies for rare diseases. The CRISPR/Cas9 system stands out as the most extensively used method for genome editing due to its relative simplicity and superior efficiency compared to TALEN and ZFN. CRISPR/Cas9 is emerging as a feasible gene-editing option to treat rare monogenic and other genetically defined human diseases. Less than 5% of ~7000 known rare diseases have FDA-approved therapies, providing a compelling need for additional research and clinical trials to identify efficient treatment options for patients with rare diseases. Development of efficient genome-editing tools capable to correct or replace dysfunctional genes will lead to novel therapeutic approaches in these diseases.

A CRISPR/Anti-CRISPR Genome Editing Approach Underlines the Synergy of Butanol Dehydrogenases in Clostridium acetobutylicum DSM 792.

Although Clostridium acetobutylicum is the model organism for the study of acetone-butanol-ethanol (ABE) fermentation, its characterization has long been impeded by the lack of efficient genome editing tools. In particular, the contribution of alcohol dehydrogenases to solventogenesis in this bacterium has mostly been studied with the generation of single-gene deletion strains. In this study, the three butanol dehydrogenase-encoding genes located on the chromosome of the DSM 792 reference strain were deleted iteratively by using a recently developed CRISPR-Cas9 tool improved by using an anti-CRISPR protein-encoding gene, acrIIA4 Although the literature has previously shown that inactivation of either bdhA, bdhB, or bdhC had only moderate effects on the strain, this study shows that clean deletion of both bdhA and bdhB strongly impaired solvent production and that a triple mutant ΔbdhA ΔbdhB ΔbdhC was even more affected. Complementation experiments confirmed the key role of these enzymes and the capacity of each bdh copy to fully restore efficient ABE fermentation in the triple deletion strain.IMPORTANCE An efficient CRISPR-Cas9 editing tool based on a previous two-plasmid system was developed for Clostridium acetobutylicum and used to investigate the contribution of chromosomal butanol dehydrogenase genes during solventogenesis. Thanks to the control of cas9 expression by inducible promoters and of Cas9-guide RNA (gRNA) complex activity by an anti-CRISPR protein, this genetic tool allows relatively fast, precise, markerless, and iterative modifications in the genome of this bacterium and potentially of other bacterial species. As an example, scarless mutants in which up to three genes coding for alcohol dehydrogenases are inactivated were then constructed and characterized through fermentation assays. The results obtained show that in C. acetobutylicum, other enzymes than the well-known AdhE1 are crucial for the synthesis of alcohol and, more globally, to perform efficient solventogenesis.

CRISPR/Cas9-Based Gene Editing Using Egg Cell-Specific Promoters in Arabidopsis and Soybean.

CRISPR/Cas9-based systems are efficient genome editing tools in a variety of plant species including soybean. Most of the gene edits in soybean plants are somatic and non-transmissible when Cas9 is expressed under control of constitutive promoters. Tremendous effort, therefore, must be spent to identify the inheritable edits occurring at lower frequencies in plants of successive generations. Here, we report the development and validation of genome editing systems in soybean and Arabidopsis based on Cas9 driven under four different egg-cell specific promoters. A soybean ubiquitin gene promoter driving expression of green fluorescent protein (GFP) is incorporated in the CRISPR/Cas9 constructs for visually selecting transgenic plants and transgene-evicted edited lines. In Arabidopsis, the four systems all produced a collection of mutations in the T2 generation at frequencies ranging from 8.3 to 42.9%, with egg cell-specific promoter AtEC1.2e1.1p being the highest. In soybean, function of the gRNAs and Cas9 expressed under control of the CaMV double 35S promoter (2x35S) in soybean hairy roots was tested prior to making stable transgenic plants. The 2x35S:Cas9 constructs yielded a high somatic mutation frequency in soybean hairy roots. In stable transgenic soybean T1 plants, AtEC1.2e1.1p:Cas9 yielded a mutation rate of 26.8%, while Cas9 expression driven by the other three egg cell-specific promoters did not produce any detected mutations. Furthermore, the mutations were inheritable in the T2 generation. Our study provides CRISPR gene-editing platforms to generate inheritable mutants of Arabidopsis and soybean without the complication of somatic mutagenesis, which can be used to characterize genes of interest in Arabidopsis and soybean.

The application of a heat-inducible CRISPR/Cas12b (C2c1) genome editing system in tetraploid cotton (G.hirsutum) plants.

SummaryThe CRISPR/Cas9 and Cas12a (Cpf1) tools have been used on a large scale for genome editing. A new effector with a single nuclease domain, a relatively small size, low‐frequency off‐target effects and cleavage capability under high temperature has been recently established and designated CRISPR/Cas12b (C2c1). Cas12b has also shown temperature inducibility in mammalian systems. Therefore, this system is potentially valuable for editing the genomes of plant species, such as cotton, that are resistant to high temperatures. Using this new system, mutants of upland cotton were successfully generated following Agrobacterium‐mediated genetic transformation under a range of temperatures. Transformants (explants infected by Agrobacterium) exposed to 45 °C for 4 days showed the highest editing efficiency. No off‐target mutation was detected by whole‐genome sequencing. Genome edits by AacCas12b in T0 generation were faithfully passed to the T1 generation. Taken together, CRISPR/Cas12b is therefore an efficient and precise tool for genome editing in cotton plants.

CRISPR-Cas12a delivery by DNA-mediated bioresponsive editing for cholesterol regulation.

CRISPR-Cas12a represents an efficient tool for genome editing in addition to the extensively investigated CRISPR-Cas9. However, development of efficient nonviral delivery system for CRISPR-Cas12a remains challenging. Here, we demonstrate a DNA nanoclew (NC)-based carrier for delivery of Cas12a/CRISPR RNA (crRNA) ribonucleoprotein (RNP) toward regulating serum cholesterol levels. The DNA NC could efficiently load the Cas12a/crRNA RNP through complementation between the DNA NC and the crRNA. Addition of a cationic polymer layer condensed the DNA-templated core and allowed further coating of a charge reversal polymer layer, which makes the assembly negatively charged under a physiological pH but reverts to positive charge under an acidic environment. When Pcsk9 was selected as the target gene because of its important role in regulating the level of serum cholesterol, efficient Pcsk9 disruption was observed in vivo (~48%), significantly reducing the expression of PCSK9 and gaining the therapeutic benefit of cholesterol control (~45% of cholesterol reduction).

Effective identification of CRISPR/Cas9-induced and naturally occurred mutations in rice using a multiplex ligation-dependent probe amplification-based method.

A multiplex ligation-dependent probe amplification (MLPA)-based method was developed and successfully utilized to efficiently detect both CRISPR/Cas9-induced and naturally occurred mutations in rice. The site-specific nuclease-based CRISPR/Cas9 system has emerged as one of the most efficient genome editing tools to modify multiple genomic targets simultaneously in various organisms, including plants for both fundamental and applied researches. Screening for both on-target and off-target mutations in CRISPR/Cas9-generated mutants at the early stages is an indispensable step for functional analysis and subsequent application. Various methods have been developed to detect CRISPR/Cas9-induced mutations in plants. Still, very few have focused on the detection of both on- and off-targets simultaneously, let alone the detection of natural mutations. Here, we report a multiplex capable method that allows to detect CRISPR/Cas9 induced on- and off-target mutations as well as naturally occurred mutation based on a multiplex ligation-dependent probe amplification (MLPA) method. We demonstrated that unlike other methods, the modified target-specific MLPA method can accurately identify any INDELs generated naturally or by the CRISPR/Cas9 system and that it can detect natural variation and zygosity of the CRISPR/Cas9-generated mutants in rice as well. Furthermore, its high sensitivity allowed to define INDELs down to 1bp and substitutions to a single nucleotide. Therefore, this sensitive, reliable, and cheap method would further accelerate functional analysis and marker-assisted breeding in plants, including rice.