Biolistic-delivery-based transient CRISPR/Cas9 expression enables in planta genome editing in wheat

Abstract

The current application of genome editing to crop plants is limited to cultivars that are amenable to in vitro culture and regeneration. Here, we report an in planta genome-editing which does not require callus culture and regeneration. Shoot apical meristems (SAMs) contain a subepidermal cell layer, L2, from which germ cells later develop during floral organogenesis. The biolistic delivery of gold particles coated with plasmids expressing CRISPR/Cas9 components designed to target TaGASR7 were bombarded into SAM-exposed embryos of imbibed seeds. Bombarded embryos showing transient GFP expression within SAM were selected and grown into adult plants. Mutations in the target gene were assessed in fifth-leaf tissue by cleaved amplified polymorphic sequence analysis. Eleven (5.2%) of the 210 bombarded plants carried mutant alleles, and the mutations of three (1.4%) of these were inherited in the next generation. Genotype analysis of T1 plants identified plants homozygous for the three homeologous genes, which were all derived from one T0 plant. These plants showed no detectable integration of the Cas9 and guide RNA genes, indicating that transient expression of CRISPR/Cas9 introduced the mutations. Together, our current method can be used to achieve in planta genome editing in wheat using CRISPR/Cas9 and suggests possible applications to other recalcitrant plant species and variations.

Perspectives of CRISPR/Cas-mediated cis-engineering in horticulture: unlocking the neglected potential for crop improvement

“…In planta transformation takes advantage of natural biological processes, which makes it a valuable alternative to in vitro tissue culture and regeneration 132,133 . Various plant cells or tissues can be the ideal transformation targets such as germline or meristematic cells 116,134,135 and dormant buds 136 . Recently, in planta particle bombardment has been used to deliver CRISPR/Cas9 DNA into shoot apical meristems, resulting in transgene-free homozygous mutated wheat plants 134 .…”

“…Various plant cells or tissues can be the ideal transformation targets such as germline or meristematic cells 116,134,135 and dormant buds 136 . Recently, in planta particle bombardment has been used to deliver CRISPR/Cas9 DNA into shoot apical meristems, resulting in transgene-free homozygous mutated wheat plants 134 . Moreover, recent efforts have been made to deliver RNPs into embryo cells in maize 135 and wheat 116 by particle bombardment and into zygotes by polyethylene glycol-Ca 2+ -mediated transfection in rice 127 .…”

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

“…CRISPR reagents can be delivered into plants by Agrobacterium mediated T-DNA transfer (Char et al 2017;Lee et al 2019), biolistic plasmid delivery (Svitashev et al 2016;Gil-Humanes et al 2017;Hamada et al 2018) or biolistic delivery of ribonucleoprotein (RNP) complexes (Svitashev et al 2016;Liang et al 2018Liang et al , 2019. Using purified Cas9 or Cas12a proteins and chemically synthesized guide RNAs eliminates the possibility of continuous expression and ensures that these reagents are present transiently and thus minimizing the opportunity for off-target editing to occur (Svitashev et al 2016).…”

Guidelines for Optimized Gene Knockout Using CRISPR/Cas9

“…Uncoupling administration of the sgRNA and Cas9 protein (e.g., in the context of genome-scale screens) can lead to successful gene editing in human primary cells [34] and appears to be more efficient upon delivery of Cas9 protein complexed with a nontargeting gRNA [35]. Finally, biolistic transfer of Cas9/sgRNA RNP complexes or of Cas9- and sgRNA-encoding plasmids appears to be an attractive alternative for cells resistant to other delivery methods, such as plant cells [36]. …”