Single and multiple gene knockouts by CRISPR–Cas9 in maize

Doll, Nicolas M.; Gilles, Laurine; Gérentes, Marie-France; Richard, Christelle; Just, Jérémy; Fierlej, Yannick; Borrelli, Virginia Maria Grazia; Gendrot, Ghislaine; Ingram, Gwyneth; Rogowsky, Peter; Widiez, Thomas

doi:10.1007/s00299-019-02378-1

Cited by 51 publications

(30 citation statements)

References 65 publications

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“…Within this pool of data, the off-target rate (Number of detected off-target effects/number of analyzed potential off-target sequences) for one mismatch appeared to be nearly 60% of the analyzed sequences, while nearly no off-target effect occurs for a sequence with at least four mismatches to further similar sequences in the genome. These findings are in line with several articles published so far (e.g., Fu et al, 2013;Hahn and Nekrasov, 2018;Doll et al, 2019). The observation that a single mismatch between on-and off-target sequences often leads to off-target effects while designing a highly specific sgRNA reduces off-target effects to a minimum allows a flexible choice of sgRNA design depending on the research question.…”

Section: Factors Affecting Off-target Effects Caused By the Use Of Crsupporting

confidence: 89%

Which Factors Affect the Occurrence of Off-Target Effects Caused by the Use of CRISPR/Cas: A Systematic Review in Plants

et al. 2020

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CRISPR/Cas enables a targeted modification of DNA sequences. Despite their ease and efficient use, one limitation is the potential occurrence of associated off-target effects. This systematic review aims to answer the following research question: Which factors affect the occurrence of off-target effects caused by the use of CRISPR/Cas in plants? Literature published until March 2019 was considered for this review. Articles were screened for relevance based on pre-defined inclusion criteria. Relevant studies were subject to critical appraisal. All studies included in the systematic review were synthesized in a narrative report, but studies rated as high and medium/high validity were reported separately from studies rated as low and medium/low or unclear validity. In addition, we ran a binary logistic regression analysis to verify five factors that may affect the occurrence of off-target effects: (1) Number of mismatches (2) Position of mismatches (3) GC-content of the targeting sequence (4) Altered nuclease variants (5) Delivery methods. In total, 180 relevant articles were included in this review containing 468 studies therein. Seventy nine percentage of these studies were rated as having high or medium/high validity. Within these studies, 6,416 potential off-target sequences were assessed for the occurrence of off-target effects. Results clearly indicate that an increased number of mismatches between the on-target and potential off-target sequence steeply decreases the likelihood of off-target effects. The observed rate of off-target effects decreased from 59% when there is one mismatch between the on-target and off-target sequences toward 0% when four or more mismatches exist. In addition, mismatch/es located within the first eight nucleotides proximal to the PAM significantly decreased the occurrence of off-target effects. There is no evidence that the GC-content significantly affects off-target effects. The database regarding the impact of the nuclease variant and the delivery method is very poor as the majority of studies applied the standard nuclease SpCas9 and the CRISPR/Cas system was stably delivered in the genome. Hence, a general significant impact of these two factors on the occurrence of off-target effects cannot be proved. This identified evidence gap needs to be filled by systematic studies exploring these individual factors in sufficient numbers.

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Section: Factors Affecting Off-target Effects Caused By the Use Of Crsupporting

confidence: 89%

Which Factors Affect the Occurrence of Off-Target Effects Caused by the Use of CRISPR/Cas: A Systematic Review in Plants

et al. 2020

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“…Our results showed that indel mutations at a single gRNA site were more common than a deletion between both sites in tobacco plants, which is consistent with previous studies in Arabidopsis, Nicotiana benthamiana, Z. mays, and O. sativa (Zhou et al, 2014;Ordon et al, 2017;Durr et al, 2018;Doll et al, 2019;Khumsupan et al, 2020). Transient assays offer a useful system to pre-select gRNA candidates before stable expression, as differences in the mutation efficiency of each gRNA can reduce the efficiency of deletions between two target sites (Zhou et al, 2014;Doll et al, 2019). However, in agreement with our findings, other studies have reported a lower frequency of large deletions in transgenic plants with gRNAs that appeared highly efficient in transient assays (Zhou et al, 2014;Khumsupan et al, 2020).…”

Section: Discussionsupporting

confidence: 93%

CRISPR-Cas9-Mediated Mutagenesis of the Rubisco Small Subunit Family in Nicotiana tabacum

et al. 2020

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Engineering the small subunit of the key CO2-fixing enzyme Rubisco (SSU, encoded by rbcS) in plants currently poses a significant challenge, as many plants have polyploid genomes and SSUs are encoded by large multigene families. Here, we used CRISPR-Cas9-mediated genome editing approach to simultaneously knock-out multiple rbcS homologs in the model tetraploid crop tobacco (Nicotiana tabacum cv. Petit Havana). The three rbcS homologs rbcS_S1a, rbcS_S1b and rbcS_T1 account for at least 80% of total rbcS expression in tobacco. In this study, two multiplexing guide RNAs (gRNAs) were designed to target homologous regions in these three genes. We generated tobacco mutant lines with indel mutations in all three genes, including one line with a 670 bp deletion in rbcS-T1. The Rubisco content of three selected mutant lines in the T1 generation was reduced by ca. 93% and mutant plants accumulated only 10% of the total biomass of wild-type plants. As a second goal, we developed a proof-of-principle approach to simultaneously introduce a non-native rbcS gene while generating the triple SSU knockout by co-transformation into a wild-type tobacco background. Our results show that CRISPR-Cas9 is a viable tool for the targeted mutagenesis of rbcS families in polyploid species and will contribute to efforts aimed at improving photosynthetic efficiency through expression of superior non-native Rubisco enzymes in plants.

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“…Choosing a competent delivery vector is important when using CRISPR/Cas9 for functional genetics. A variety of strategies and vectors have been successfully used in maize [8][9][10], and each may have benefits. A custom vector modified from pRGEB32 (gifted by Yinong Yang) for use in maize is described here.…”

Section: Considerations For Experimental Designmentioning

confidence: 99%

CRISPR/Cas9 Targeted Mutagenesis for Functional Genetics in Maize

Hunter

2021

Plants

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The CRISPR/Cas9-based system for targeted mutagenesis has become an indispensable tool for functional genetics in plants. CRISPR/Cas9 allows users to generate loss-of-function alleles in genes of interest with precision and in a simple-to-use system. This manuscript outlines important points to consider for experimental design and utilization of CRISPR/Cas9 in targeted mutagenesis in maize. It also introduces the pRGEB32-BAR vector modified for use in maize that allows simultaneous delivery of multiple gRNAs using a simple assembly. Vector selection, gRNA design, genetic strategies, and genotyping approaches are discussed, with an emphasis on achieving isolation of homozygous mutant plants in a time- and cost-efficient manner.

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Single and multiple gene knockouts by CRISPR–Cas9 in maize

Abstract: The analysis of 93 mutant alleles in 18 genes demonstrated that CRISPR-Cas9 is a robust tool for targeted mutagenesis in maize, permitting efficient generation of single and multiple knockouts.

Cited by 51 publications

References 65 publications

Which Factors Affect the Occurrence of Off-Target Effects Caused by the Use of CRISPR/Cas: A Systematic Review in Plants

Which Factors Affect the Occurrence of Off-Target Effects Caused by the Use of CRISPR/Cas: A Systematic Review in Plants

CRISPR-Cas9-Mediated Mutagenesis of the Rubisco Small Subunit Family in Nicotiana tabacum

CRISPR/Cas9 Targeted Mutagenesis for Functional Genetics in Maize

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