Comparison of CRISPR-Cas9/Cas12a Ribonucleoprotein Complexes for Genome Editing Efficiency in the Rice Phytoene Desaturase (OsPDS) Gene

Banakar, Raviraj; Schubert, Mollie S.; Collingwood, Michael A.; Vakulskas, Christopher A.; Eggenberger, Alan L.; Wang, Kan

doi:10.1186/s12284-019-0365-z

Cited by 84 publications

(55 citation statements)

References 31 publications

(33 reference statements)

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“…CRISPR/Cas editing is regarded as fairly specific in plants assuming stringent target selection (Hahn & Nekrasov, 2019), but efficiency may be increased for certain Cas proteins, depending on the organism to be edited ( Table 1). In rice, off-target edits by both Cas9 and Cas12a appear to rarely occur, but Cas12a may be more efficient for on-target edits (Yin et al, 2017, Tang et al, 2018, Banakar et al, 2020. However, in maize, SpCas9 was more efficient for on-target edits, with no detectable off-target mutations identified in plants from the generation after editing (Lee et al, 2019).…”

Section: Off-target Considerationsmentioning

confidence: 96%

Current Capabilities, Precision, and Risks of Genome Editing Techniques in Agricultural Systems

Carter¹

2020

Preprint

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We are in a new chapter of crop and livestock improvement with the emergence of genome editing. This latest generation of molecular tools can be used to make targeted changes in a genome including insertions, deletions, and mutations. With new advances comes new risks for unintended changes and impacts, thus the need for appropriate risk assessment for product development and to inform regulatory measures. Though CRISPR/Cas has arisen as the predominant technology, there are multiple types of genome editing tools each with pros and cons depending on the organism and desired outcome. Furthermore, each editing tool differs in specificity as they may edit non-intended sites, referred to as off-target edits. The consensus of the agricultural editing community is to avoid off-target editing through design and detection, instead of determining whether off-target editing in each case is detrimental. The design of a targeting component, the tool chosen, and the identification of the edit(s) made are the critical factors in avoiding off-target edits and confirming intended edits in final products that are released commercially. The limited amount of head-to-head comparisons of genome editing tools in diverse crops and livestock make it difficult to develop broad conclusions and best practices, which is further compounded by the diversity of techniques, targets, and processes. Developers and breeders should consult the literature and test as needed to determine which editing technology will be the most effective for their purposes, especially as more tools with altered efficiency and specificity become available. Yet, the lack of off-target edits in studies that employed careful design of targeting components followed by wide testing for on- and off-target edits bodes well for the use of genome editing with proper precautions of target selection and screening.

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Section: Off-target Considerationsmentioning

confidence: 96%

Current Capabilities, Precision, and Risks of Genome Editing Techniques in Agricultural Systems

Carter¹

2020

Preprint

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“…Utilizing the LbCas12a nuclease two endogenous rice genes OsPDS and OsBEL were targeted with mutation frequencies of 21.4 and 41.2%, respectively ( Xu et al, 2017 ). An independent study that targeted the disruption of OsPDS by LbCas12a resulted in a similarly high editing frequency of 32.3% ( Banakar et al, 2020 ). Xu et al, 2017 also demonstrated that pre-crRNAs were more efficient in generating mutants than mature crRNAs in rice.…”

Section: Application Of Crispr-cas12a In Agriculturementioning

confidence: 98%

“…Cas12a editing has been widely utilized in many crops (see Table 1 ) including rice ( Endo A. et al, 2016 ; Begemann et al, 2017 ; Hu et al, 2017 ; Tang et al, 2017 , 2018 ; Wang et al, 2017a , b ; Yin et al, 2017 ; Li L. et al, 2018 ; Li et al, 2019a ; Jun et al, 2019 ; Malzahn et al, 2019 ; Banakar et al, 2020 ; Chen et al, 2020 ; Schindele and Puchta, 2020 ), wheat ( Liu et al, 2020 ), maize ( Lee K. et al, 2019 ), soybean ( Kim et al, 2017 ), cotton ( Li B. et al, 2019 ), tomato ( van Vu et al, 2020 ), citrus ( Jia et al, 2019 ), tobacco ( Endo A. et al, 2016 ; Endo and Toki, 2019 ), and the model plant Arabidopsis ( Wolter and Puchta, 2019 ; Schindele and Puchta, 2020 ). At present, three Cas12a genome editing systems AsCas12a, FnCas12a, and LbCas12a have been demonstrated in plants ( Zhong et al, 2018 ) with varied efficiency.…”

Section: Application Of Crispr-cas12a In Agriculturementioning

confidence: 99%

“…Given the inherent variations in site-specific editing efficiencies, it is challenging to directly compare the mutation efficiencies of Cas9 and Cas12a. Although studies in several plant species have suggested lower editing efficiencies associated with Cas12a relative to Cas9 ( Lee K. et al, 2019 ; Malzahn et al, 2019 ; Liu et al, 2020 ), Wang and colleagues used Cas9 and Cas12a to target the same loci and in one instance observed a higher efficiency of mutation with Cas9 ( Lee K. et al, 2019 ) and with another target Cas12a was more efficient ( Banakar et al, 2020 ). Various factors which might have attributed to the relative efficiency could be related to the gRNA sequences, epigenetic modifications of the target site or expression of the endonuclease itself.…”

Section: Application Of Crispr-cas12a In Agriculturementioning

confidence: 99%

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CRISPR-Cas12a (Cpf1): A Versatile Tool in the Plant Genome Editing Tool Box for Agricultural Advancement

et al. 2020

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Global population is predicted to approach 10 billion by 2050, an increase of over 2 billion from today. To meet the demands of growing, geographically and socio-economically diversified nations, we need to diversity and expand agricultural production. This expansion of agricultural productivity will need to occur under increasing biotic, and environmental constraints driven by climate change. Clustered regularly interspaced short palindromic repeats-site directed nucleases (CRISPR-SDN) and similar genome editing technologies will likely be key enablers to meet future agricultural needs. While the application of CRISPR-Cas9 mediated genome editing has led the way, the use of CRISPR-Cas12a is also increasing significantly for genome engineering of plants. The popularity of the CRISPR-Cas12a, the type V (class-II) system, is gaining momentum because of its versatility and simplified features. These include the use of a small guide RNA devoid of trans-activating crispr RNA, targeting of T-rich regions of the genome where Cas9 is not suitable for use, RNA processing capability facilitating simpler multiplexing, and its ability to generate double strand breaks (DSB) with staggered ends. Many monocot and dicot species have been successfully edited using this Cas12a system and further research is ongoing to improve its efficiency in plants, including improving the temperature stability of the Cas12a enzyme, identifying new variants of Cas12a or synthetically producing Cas12a with flexible PAM sequences. In this review we provide a comparative survey of CRISPR-Cas12a and Cas9, and provide a perspective on applications of CRISPR-Cas12 in agriculture.

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“…We first applied the double-barrel/CellProfiler™ (DB-CP) system to evaluate the effectiveness of transfection reagent TransIT ® -2020 (Mirus Bio LLC, Madison, WI, USA) for the literature used spermidine as the standard reagent to precipitate DNA onto gold particles [5,23,27] . In recent years, however, TransIT ® -2020 has emerged as an alternative reagent for introducing DNA [28] as well as ribonucleoprotein [29][30][31] into plant tissues. Interestingly, despite its successful application in plants, there has been scant information for optimizing the coating of DNA onto gold with the TransIT ® -2020.…”

Section: Application Of the Double-barrel Cellprofiler™ Systemmentioning

confidence: 99%

An improved biolistic delivery and analysis method for evaluation of DNA and CRISPR-Cas delivery efficacy in plant tissue

Miller

Eggenberger

Lee

et al. 2020

Preprint

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Biolistic delivery is widely used for genetic transformation but inconsistency between bombardment samples for transient gene expression analysis often hinders quantitative analyses. We developed a methodology to improve the consistency of biolistic delivery results by using a double-barrel device and a cell counting software. The double-barrel device enables a strategy of incorporating an internal control into each sample, which significantly decreases variance of the results. The cell counting software further reduces errors and increases throughput. The utility of this new platform is demonstrated by optimizing conditions for delivering DNA using the commercial transfection reagent TransIT®-2020. In addition, the same approach is applied to test the efficacy of CRISPR-Cas9-mediated gene editing. The novel combination of the bombardment device and analysis method allows simultaneous comparison and optimization of parameters in the biolistic delivery. The platform developed here can be broadly applied to any target samples using biolistics, including animal cells and tissues.

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Comparison of CRISPR-Cas9/Cas12a Ribonucleoprotein Complexes for Genome Editing Efficiency in the Rice Phytoene Desaturase (OsPDS) Gene

Cited by 84 publications

References 31 publications

Current Capabilities, Precision, and Risks of Genome Editing Techniques in Agricultural Systems

Current Capabilities, Precision, and Risks of Genome Editing Techniques in Agricultural Systems

CRISPR-Cas12a (Cpf1): A Versatile Tool in the Plant Genome Editing Tool Box for Agricultural Advancement

An improved biolistic delivery and analysis method for evaluation of DNA and CRISPR-Cas delivery efficacy in plant tissue

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