Genome editing promises giant leaps forward in advancing biotechnology, agriculture, and basic research. The process relies on the use of sequence specific nucleases (SSNs) to make DNA double ...stranded breaks at user defined genomic loci, which are subsequently repaired by two main DNA repair pathways: non-homologous end joining (NHEJ) and homology directed repair (HDR). NHEJ can result in frameshift mutations that often create genetic knockouts. These knockout lines are useful for functional and reverse genetic studies but also have applications in agriculture. HDR has a variety of applications as it can be used for gene replacement, gene stacking, and for creating various fusion proteins. In recent years, transcription activator-like effector nucleases and clustered regularly interspaced palindromic repeats (CRISPR) and CRISPR associated protein 9 or CRISPR from Prevotella and Francisella 1 have emerged as the preferred SSNs for research purposes. Here, we review their applications in plant research, discuss current limitations, and predict future research directions in plant genome editing.
The application of clustered regularly interspaced short palindromic repeats (CRISPR) for genetic manipulation has revolutionized life science over the past few years. CRISPR was first discovered as ...an adaptive immune system in bacteria and archaea, and then engineered to generate targeted DNA breaks in living cells and organisms. During the cellular DNA repair process, various DNA changes can be introduced. The diverse and expanding CRISPR toolbox allows programmable genome editing, epigenome editing and transcriptome regulation in plants. However, challenges in plant genome editing need to be fully appreciated and solutions explored. This Review intends to provide an informative summary of the latest developments and breakthroughs of CRISPR technology, with a focus on achievements and potential utility in plant biology. Ultimately, CRISPR will not only facilitate basic research, but also accelerate plant breeding and germplasm development. The application of CRISPR to improve germplasm is particularly important in the context of global climate change as well as in the face of current agricultural, environmental and ecological challenges.
Targeting specificity has been a barrier to applying genome editing systems in functional genomics, precise medicine and plant breeding. In plants, only limited studies have used whole-genome ...sequencing (WGS) to test off-target effects of Cas9. The cause of numerous discovered mutations is still controversial. Furthermore, WGS-based off-target analysis of Cpf1 (Cas12a) has not been reported in any higher organism to date.
We conduct a WGS analysis of 34 plants edited by Cas9 and 15 plants edited by Cpf1 in T0 and T1 generations along with 20 diverse control plants in rice. The sequencing depths range from 45× to 105× with read mapping rates above 96%. Our results clearly show that most mutations in edited plants are created by the tissue culture process, which causes approximately 102 to 148 single nucleotide variations (SNVs) and approximately 32 to 83 insertions/deletions (indels) per plant. Among 12 Cas9 single guide RNAs (sgRNAs) and three Cpf1 CRISPR RNAs (crRNAs) assessed by WGS, only one Cas9 sgRNA resulted in off-target mutations in T0 lines at sites predicted by computer programs. Moreover, we cannot find evidence for bona fide off-target mutations due to continued expression of Cas9 or Cpf1 with guide RNAs in T1 generation.
Our comprehensive and rigorous analysis of WGS data across multiple sample types suggests both Cas9 and Cpf1 nucleases are very specific in generating targeted DNA modifications and off-targeting can be avoided by designing guide RNAs with high specificity.
CRISPR-Cas12a is a promising genome editing system for targeting AT-rich genomic regions. Comprehensive genome engineering requires simultaneous targeting of multiple genes at defined locations. ...Here, to expand the targeting scope of Cas12a, we screen nine Cas12a orthologs that have not been demonstrated in plants, and identify six, ErCas12a, Lb5Cas12a, BsCas12a, Mb2Cas12a, TsCas12a and MbCas12a, that possess high editing activity in rice. Among them, Mb2Cas12a stands out with high editing efficiency and tolerance to low temperature. An engineered Mb2Cas12a-RVRR variant enables editing with more relaxed PAM requirements in rice, yielding two times higher genome coverage than the wild type SpCas9. To enable large-scale genome engineering, we compare 12 multiplexed Cas12a systems and identify a potent system that exhibits nearly 100% biallelic editing efficiency with the ability to target as many as 16 sites in rice. This is the highest level of multiplex edits in plants to date using Cas12a. Two compact single transcript unit CRISPR-Cas12a interference systems are also developed for multi-gene repression in rice and Arabidopsis. This study greatly expands the targeting scope of Cas12a for crop genome engineering.
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The relative ease, speed, and biological scope of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated Protein9 (Cas9)-based reagents for genomic manipulations are ...revolutionizing virtually all areas of molecular biosciences, including functional genomics, genetics, applied biomedical research, and agricultural biotechnology. In plant systems, however, a number of hurdles currently exist that limit this technology from reaching its full potential. For example, significant plant molecular biology expertise and effort is still required to generate functional expression constructs that allow simultaneous editing, and especially transcriptional regulation, of multiple different genomic loci or multiplexing, which is a significant advantage of CRISPR/Cas9 versus other genome-editing systems. To streamline and facilitate rapid and wide-scale use of CRISPR/Cas9-based technologies for plant research, we developed and implemented a comprehensive molecular toolbox for multifaceted CRISPR/Cas9 applications in plants. This toolbox provides researchers with a protocol and reagents to quickly and efficiently assemble functional CRISPR/Cas9 transfer DNA constructs for monocots and dicots using Golden Gate and Gateway cloning methods. It comes with a full suite of capabilities, including multiplexed gene editing and transcriptional activation or repression of plant endogenous genes. We report the functionality and effectiveness of this toolbox in model plants such as tobacco (Nicotiana benthamiana), Arabidopsis (Arabidopsis thaliana), and rice (Oryza sativa), demonstrating its utility for basic and applied plant research.
CRISPR-derived biotechnologies have revolutionized the genetic engineering field and have been widely applied in basic plant research and crop improvement. Commonly used Agrobacterium- or particle ...bombardment-mediated transformation approaches for the delivery of plasmid-encoded CRISPR reagents can result in the integration of exogenous recombinant DNA and potential off-target mutagenesis. Editing efficiency is also highly dependent on the design of the expression cassette and its genomic insertion site. Genetic engineering using CRISPR ribonucleoproteins (RNPs) has become an attractive approach with many advantages: DNA/transgene-free editing, minimal off-target effects, and reduced toxicity due to the rapid degradation of RNPs and the ability to titrate their dosage while maintaining high editing efficiency. Although RNP-mediated genetic engineering has been demonstrated in many plant species, its editing efficiency remains modest, and its application in many species is limited by difficulties in plant regeneration and selection. In this review, we summarize current developments and challenges in RNP-mediated genetic engineering of plants and provide future research directions to broaden the use of this technology.
CRISPR-derived genome engineering technologies have been widely applied in plant science. Delivery of CRISPR reagents as RNPs is a promising approach for the generation of transgene-free genome-edited crops. This review summarizes recent progress, discusses present challenges, and presents some future perspectives on this topic.
Advanced CRISPR-Cas9 based technologies first validated in mammalian cell systems are quickly being adapted for use in plants. These new technologies increase CRISPR-Cas9's utility and effectiveness ...by diversifying cellular capabilities through expression construct system evolution and enzyme orthogonality, as well as enhanced efficiency through delivery and expression mechanisms. Here, we review the current state of advanced CRISPR-Cas9 and Cpf1 capabilities in plants and cover the rapid evolution of these tools from first generation inducers of double strand breaks for basic genetic manipulations to second and third generation multiplexed systems with myriad functionalities, capabilities, and specialized applications. We offer perspective on how to utilize these tools for currently untested research endeavors and analyze strengths and weaknesses of novel CRISPR systems in plants. Advanced CRISPR functionalities and delivery options demonstrated in plants are primarily reviewed but new technologies just coming to the forefront of CRISPR development, or those on the horizon, are briefly discussed. Topics covered are focused on the expansion of expression and delivery capabilities for CRISPR-Cas9 components and broadening targeting range through orthogonal Cas9 and Cpf1 proteins.
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