Improved CRISPR/Cas9 gene editing by fluorescence activated cell sorting of green fluorescence protein tagged protoplasts

Petersen, Bent Larsen; Møller, Svenning Rune; Mravec, Jozef; Jørgensen, Bodil; Christensen, Mikkel A.; Liu, Ying; Wandall, Hans H.; Bennett, Eric; Yang, Zifeng

doi:10.1186/s12896-019-0530-x

Cited by 25 publications

(20 citation statements)

References 64 publications

(95 reference statements)

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“…During the preparation of the manuscript, the 2A peptide was reported to be used for fusing Cas9 and GFP in the protoplasts of Nicotiana benthamiana. Fluorescence-activated cell sorting (FACS)-mediated selection of GFPexpressed protoplasts has been shown to facilitate Cas9mediated mutation enrichment (Petersen et al 2019), which is consistent with our study showing that Cas9-P2A-GFP improves the selection of transgenic plants with a high editing efficiency. In addition to the Cas9-P2A-GFP construct used in this study, the P2A peptide may be widely used to coexpress multiple proteins in plants for various purposes.…”

Section: Coexpression Of Multiple Proteins Using 2a Peptidessupporting

confidence: 91%

A novel CRISPR/Cas9 system for efficiently generating Cas9-free multiplex mutants in Arabidopsis

Wang

Chen

2019

aBIOTECH

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The CRISPR/Cas9 genome-editing system has emerged as a popular powerful tool for biological research. However, the process of selecting efficiently edited Cas9-free plants is usually laborious and time consuming. Here, we demonstrated P2A to be the most efficient self-cleaving peptide for fusing Cas9 and GFP in Arabidopsis and then used Cas9-P2A-GFP to develop a novel CRISPR/Cas9 system. Additionally, a pair of isocaudomer restriction enzymes were selected to conveniently assemble multiple sgRNAs. In this system, the GFP fluorescence intensity in T1 transgenic plants indicates the expression level of the Cas9 protein, which correlates well with the editing efficiency. Furthermore, Cas9-free plants can be easily selected by examining GFP fluorescence in T2 transgenic plants. The efficient knockout of BRI1, BZR1 and BES1 demonstrated the robustness of our new system. Thus, we designed a novel CRISPR/Cas9 system that can generate Cas9-free multiplex mutants efficiently in Arabidopsis and possibly in other plant species.

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Section: Coexpression Of Multiple Proteins Using 2a Peptidessupporting

confidence: 91%

A novel CRISPR/Cas9 system for efficiently generating Cas9-free multiplex mutants in Arabidopsis

Wang

Chen

2019

aBIOTECH

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“…The Indel Amplicon Analysis (IDAA) 10 technique allows for fast and direct assessment of insertions/deletions (indels), with a sensitivity down to + /−1 bp, without the need for in depth Sanger sequencing 11 . IDAA was recently used for editing scoring of protoplast cell populations 9 , and in the present study the use of IDAA was expanded and adapted to plants with complex genomes, such as potato, where it proved an efficient and fast tool for editing assessment.…”

Section: Introductionmentioning

confidence: 98%

“…High efficient CRISPR/Cas9 editing are in many plants complicated by the presence of complex and high ploidy genomes and inefficient or poorly controlled delivery of the CRISPR/Cas9 components to cells with regenerative potential. Fluorescence Activated Cell Sorting (FACS) of cells expressing GFP tagged CRISPR/Cas9 is regularly used for enrichment of edited cell populations in mammalian cell systems 8 and more recently also expanded to plant protoplast cells 9 . The Indel Amplicon Analysis (IDAA) 10 technique allows for fast and direct assessment of insertions/deletions (indels), with a sensitivity down to + /−1 bp, without the need for in depth Sanger sequencing 11 .…”

Section: Introductionmentioning

confidence: 99%

High efficacy full allelic CRISPR/Cas9 gene editing in tetraploid potato

Johansen

Liu

Jørgensen

et al. 2019

Sci Rep

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CRISPR/Cas9 editing efficacies in tetraploid potato were highly improved through the use of endogenous potato U6 promoters. Highly increased editing efficiencies in the Granular Bound Starch Synthase gene at the protoplast level were obtained by replacement of the Arabidopsis U6 promotor, driving expression of the CRISPR component, with endogenous potato U6 promotors. This translated at the ex-plant level into 35% full allelic gene editing. Indel Detection Amplicon Analysis was established as an efficient tool for fast assessment of gene editing in complex genomes, such as potato. Together, this warrants significant reduction of laborious cell culturing, ex-plant regeneration and screening procedures of plants with high complexity genomes.

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“…This is illustrated by editing of the granule bound starch synthase gene GBSS in Solanum tuberosum (potato) (Figure 6A , B ), a tetraploid organism, where GBSS furthermore is represented by three allelic variants. IDAA on wild-type protoplasts ( 144 , 145 ) can reveal the presence of the four alleles, as they can be distinguished by size in the GBSS gRNA target region (Figure 6C , upper panel). Editing of potato was achieved through delivery of CRISPR/Cas9 to protoplasts that were subsequently analysed by IDAA, which revealed that indels had been induced (Figure 6C , middle panel).…”

Section: Introductionmentioning

confidence: 99%

INDEL detection, the ‘Achilles heel’ of precise genome editing: a survey of methods for accurate profiling of gene editing induced indels

Bennett

Petersen

Johansen

et al. 2020

Nucleic Acids Research

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Advances in genome editing technologies have enabled manipulation of genomes at the single base level. These technologies are based on programmable nucleases (PNs) that include meganucleases, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated 9 (Cas9) nucleases and have given researchers the ability to delete, insert or replace genomic DNA in cells, tissues and whole organisms. The great flexibility in re-designing the genomic target specificity of PNs has vastly expanded the scope of gene editing applications in life science, and shows great promise for development of the next generation gene therapies. PN technologies share the principle of inducing a DNA double-strand break (DSB) at a user-specified site in the genome, followed by cellular repair of the induced DSB. PN-elicited DSBs are mainly repaired by the non-homologous end joining (NHEJ) and the microhomology-mediated end joining (MMEJ) pathways, which can elicit a variety of small insertion or deletion (indel) mutations. If indels are elicited in a protein coding sequence and shift the reading frame, targeted gene knock out (KO) can readily be achieved using either of the available PNs. Despite the ease by which gene inactivation in principle can be achieved, in practice, successful KO is not only determined by the efficiency of NHEJ and MMEJ repair; it also depends on the design and properties of the PN utilized, delivery format chosen, the preferred indel repair outcomes at the targeted site, the chromatin state of the target site and the relative activities of the repair pathways in the edited cells. These variables preclude accurate prediction of the nature and frequency of PN induced indels. A key step of any gene KO experiment therefore becomes the detection, characterization and quantification of the indel(s) induced at the targeted genomic site in cells, tissues or whole organisms. In this survey, we briefly review naturally occurring indels and their detection. Next, we review the methods that have been developed for detection of PN-induced indels. We briefly outline the experimental steps and describe the pros and cons of the various methods to help users decide a suitable method for their editing application. We highlight recent advances that enable accurate and sensitive quantification of indel events in cells regardless of their genome complexity, turning a complex pool of different indel events into informative indel profiles. Finally, we review what has been learned about PN-elicited indel formation through the use of the new methods and how this insight is helping to further advance the genome editing field.

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Improved CRISPR/Cas9 gene editing by fluorescence activated cell sorting of green fluorescence protein tagged protoplasts

Cited by 25 publications

References 64 publications

A novel CRISPR/Cas9 system for efficiently generating Cas9-free multiplex mutants in Arabidopsis

A novel CRISPR/Cas9 system for efficiently generating Cas9-free multiplex mutants in Arabidopsis

High efficacy full allelic CRISPR/Cas9 gene editing in tetraploid potato

INDEL detection, the ‘Achilles heel’ of precise genome editing: a survey of methods for accurate profiling of gene editing induced indels

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