Proxies of CRISPR-Cas9 activity to aid in the identification of mutagenized Arabidopsis plants

Li, Renyu; Vavrik, Charles; Danna, Cristian H.

doi:10.1101/637314

Cited by 4 publications

(6 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It has been well documented that mutagenic activity differs between individual primary transformants, and that editing at a primary locus increases the likelihood of editing at further loci (e.g., Bollier et al , 2020; Li et al , 2020). Similarly, we detected mutant phenotypes at higher frequencies in families that were already scored as er gl1 double mutants in the T 1 generation (Table S2).…”

Section: Discussionmentioning

confidence: 99%

“…In a second round of analysis, primary transformants with high mutagenic activity can be pre‐selected based on the presence of mutations in one or several of these key loci. Analysis of T 2 segregants will likely allow straightforward isolation of the desired genotype, as cleavage of targets of inefficient sgRNAs shall function as an efficient marker for co‐editing of remaining sites (Li et al , 2020, see also Symeonidi et al , 2020).…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Highly efficient multiplex editing: one‐shot generation of 8× Nicotiana benthamiana and 12× Arabidopsis mutants

et al. 2021

View full text Add to dashboard Cite

SUMMARY Genome editing by RNA‐guided nucleases, such as SpCas9, has been used in numerous different plant species. However, to what extent multiple independent loci can be targeted simultaneously by multiplexing has not been well documented. Here, we developed a toolkit, based on a highly intron‐optimized zCas9i gene, which allows assembly of nuclease constructs expressing up to 32 single guide RNAs (sgRNAs). We used this toolkit to explore the limits of multiplexing in two major model species, and report on the isolation of transgene‐free octuple (8×) Nicotiana benthamiana and duodecuple (12×) Arabidopsis thaliana mutant lines in a single generation (T1 and T2, respectively). We developed novel counter‐selection markers for N. benthamiana, most importantly Sl‐FAST2, comparable to the well‐established Arabidopsis seed fluorescence marker, and FCY‐UPP, based on the production of toxic 5‐fluorouracil in the presence of a precursor. Targeting eight genes with an array of nine different sgRNAs and relying on FCY‐UPP for selection of non‐transgenic T1, we identified N. benthamiana mutant lines with astonishingly high efficiencies: All analyzed plants carried mutations in all genes (approximately 112/116 target sites edited). Furthermore, we targeted 12 genes by an array of 24 sgRNAs in A. thaliana. Efficiency was significantly lower in A. thaliana, and our results indicate Cas9 availability is the limiting factor in such higher‐order multiplexing applications. We identified a duodecuple mutant line by a combination of phenotypic screening and amplicon sequencing. The resources and results presented provide new perspectives for how multiplexing can be used to generate complex genotypes or to functionally interrogate groups of candidate genes.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Highly efficient multiplex editing: one‐shot generation of 8× Nicotiana benthamiana and 12× Arabidopsis mutants

et al. 2021

View full text Add to dashboard Cite

show abstract

“…It has been well-documented that mutagenic activity differs between individual primary transformants, and that editing at a primary locus increases the likelihood of editing at further loci (e.g., Bollier et al , Li et al , 2020). Similarly, we detected mutant phenotypes at higher frequencies in families that were already scored as er gl1 double mutants in the T 1 generation (Table S2).…”

Section: Discussionmentioning

confidence: 99%

“…In a second round of analysis, primary transformants with high mutagenic activity can be pre-selected based on presence of mutations in one or several of these key loci. Analysis of T 2 segregants will likely allow straightforward isolation of the desired genotype, as cleavage of targets of inefficient sgRNAs shall function as an efficient marker for co-editing of remaining sites (Li et al , 2020, see also Symeonidi et al , 2020).…”

Section: Discussionmentioning

confidence: 99%

Highly efficient multiplex editing: One-shot generation of 8xNicotiana benthamianaand 12x Arabidopsis mutants

Stuttmann

Barthel

Martin

et al. 2020

Preprint

View full text Add to dashboard Cite

SummaryGenome editing by RNA-guided nucleases in model species is still hampered by low efficiencies, and isolation of transgene-free individuals often requires tedious PCR screening. Here, we present a toolkit that mitigates these drawbacks for Nicotiana benthamiana and Arabidopsis thaliana. The toolkit is based on an intron-optimized SpCas9-coding gene (zCas9i), which conveys dramatically enhanced editing efficiencies. The zCas9i gene is combined with remaining components of the genome editing system in recipient vectors, which lack only the user-defined guide RNA transcriptional units. Up to 32 guide RNA transcriptional units can be introduced to these recipients by a simple and PCR-free cloning strategy, with the choice of three different RNA polymerase III promoters for guide RNA expression. We developed new markers to aid transgene counter-selection in N. benthamiana, and demonstrate their efficacy for isolation of several genome-edited N. benthamiana lines. In Arabidopsis, we explore the limits of multiplexing by simultaneously targeting 12 genes by 24 sgRNAs. Perhaps surprisingly, the limiting factor in such higher order multiplexing applications is Cas9 availability, rather than recombination or silencing of repetitive sgRNA TU arrays. Through a combination of phenotypic screening and pooled amplicon sequencing, we identify transgene-free duodecuple mutant Arabidopsis plants directly in the T2 generation. This demonstrates high efficiency of the zCas9i gene, and reveals new perspectives for multiplexing to target gene families and to generate higher order mutants.

show abstract

“…As mutation efficiencies over all six loci are correlated, we suggest the use of a target gene with an easy-to-score, nondetrimental loss-of-function phenotype as a proxy for highly mutagenized lines. As an alternative to endogenous genes (Li et al ., 2020), loss of GFP in a reporter line can also be used as one such proxy. We foresee this approach to be a powerful tool to dissect genetic networks in model and crops species alike.…”

Section: Mainmentioning

confidence: 99%

Efficient simultaneous mutagenesis of multiple genes in specific plant tissues by multiplex CRISPR

Bollier

Buono

Jacobs

2020

Preprint

View full text Add to dashboard Cite

Multiplex CRISPR approaches enable mutating multiple genes in plants, however it is unclear how feasible this is in tissue-specific mutagenesis. Here we simultaneously mutated six genes either ubiquitously or exclusively in the root cap of Arabidopsis. The mutation frequencies for all target genes were positively correlated and unaffected by the order of gRNAs in the vector, indicating that efficient higher-order mutagenesis in specific plant tissues can be readily achieved.

show abstract

Proxies of CRISPR-Cas9 activity to aid in the identification of mutagenized Arabidopsis plants

Cited by 4 publications

References 38 publications

Highly efficient multiplex editing: one‐shot generation of 8× Nicotiana benthamiana and 12× Arabidopsis mutants

Highly efficient multiplex editing: one‐shot generation of 8× Nicotiana benthamiana and 12× Arabidopsis mutants

Highly efficient multiplex editing: One-shot generation of 8xNicotiana benthamianaand 12x Arabidopsis mutants

Efficient simultaneous mutagenesis of multiple genes in specific plant tissues by multiplex CRISPR

Contact Info

Product

Resources

About