Conversion of a normal maize hybrid into a waxy version using in vivo CRISPR/Cas9 targeted mutation activity

Waxy maize is a specialty maize that produces mainly amylopectin starch with special food or industrial values. The objective of this study was to overcome the limitations of wx mutant allele acquisition and breeding efficiency by conversion of parental lines from normal to waxy maize. The intended mutation activity was achieved by in vivo CRISPR/Cas9 machinery involving desired-target mutation of the Wx locus in the ZC01 background,abbreviated as ZC01-DTM^(wx). Triple selection was applied to segregants to obtain high genome background recovery with transgene-free wx mutations. The targeted mutation was identified, yielding six types of mutations among progeny crossed with ZC01-DTM^(wx).The amylopectin contents of the endosperm starch in mutant lines and hybrids averaged94.9%, while those of the wild-type controls were significantly(P < 0.01) lower, with an average of 76.9%. Double selection in transgene-free lines was applied using the Bar strip test and Cas9 PCR screening. The genome background recovery ratios of the lines were determined using genome-wide SNP data. That of lines used as male parents was as high as98.19% and that of lines used as female parents was as high as 86.78%. Conversion hybrids and both parental lines showed agronomic performance similar to that of their wild-type counterparts. This study provides a practical example of the efficient extension of CRISPR/Cas9 targeted mutation to industrial hybrids for transformation of a recalcitrant species.

An inducible CRISPR activation tool for accelerating plant regeneration

The inducible CRISPR activation(CRISPR-a)system offers unparalleled precision and versatility for regu-lating endogenous genes,making it highly sought after in plant research.In this study,we developed a chem-ically inducible CRISPR-a tool for plants called ER-Tag by combining the LexA-VP16-ER inducible system with the SunTag CRISPR-a system.We systematically compared different induction strategies and achieved high efficiency in target gene activation.We demonstrated that guide RNAs can be multiplexed and pooled for large-scale screening of effective morphogenic genes and gene pairs involved in plant regeneration.Further experiments showed that induced activation of these morphogenic genes can accelerate regenera-tion and improve regeneration efficiency in both eudicot and monocot plants,including alfalfa,woodland strawberry,and sheepgrass.Our study expands the CRISPR toolset in plants and provides a powerful new strategy for studying gene function when constitutive expression is not feasible or ideal.

Fundamental CRISPR-Cas9 tools and current applications in microbial systems

Derived from the bacterial adaptive immune system,CRISPR technology has revolutionized conventional genetic engineering methods and unprecedentedly facilitated strain engineering.In this review,we outline the fundamental CRISPR tools that have been employed for strain optimization.These tools include CRISPR editing,CRISPR interference,CRISPR activation and protein imaging.To further characterize the CRISPR technology,we present current applications of these tools in microbial systems,including model-and non-model industrial microorganisms.Specially,we point out the major challenges of the CRISPR tools when utilized for multiplex genome editing and sophisticated expression regulation.To address these challenges,we came up with strategies that place emphasis on the amelioration of DNA repair efficiency through CRISPR-Cas9-assisted recombineering.Lastly,multiple promising research directions were proposed,mainly focusing on CRISPR-based construction of microbial ecosystems toward high production of desired chemicals.