Development of germ‐line‐specific <scp>CRISPR</scp>‐Cas9 systems to improve the production of heritable gene modifications in <i>Arabidopsis</i>

TLDR

CRISPR/Cas9 is widely used in plants, comprising a guide RNA and Cas9 nuclease, but ubiquitous expression only transmits edits from reproductive cells to progeny. The study designs and characterizes a germ‑line‑specific Cas9 system (GSC) targeting male gametocytes in Arabidopsis using an SPL expression cassette. The authors compared the GSC to a ubiquitous Cas9 system by targeting four loci in two genes, and also tested two additional germ‑line‑specific promoter systems, one of which produced heritable homozygous T1 mutants. In T2 plants, the GSC system yielded 30% mutations, produced only 29% chimeras versus 70% for the ubiquitous system, and 70% of mutants were heterozygous, with a 37% higher rate of heritable mutations and greater polymorphism, indicating that GSC will aid screening for lethal mutations in the T2 generation.

Abstract

Summary The Streptococcus ‐derived CRISPR /Cas9 system is being widely used to perform targeted gene modifications in plants. This customized endonuclease system has two components, the single‐guide RNA (sg RNA ) for target DNA recognition and the CRISPR ‐associated protein 9 (Cas9) for DNA cleavage. Ubiquitously expressed CRISPR /Cas9 systems ( UC ) generate targeted gene modifications with high efficiency but only those produced in reproductive cells are transmitted to the next generation. We report the design and characterization of a germ‐line‐specific Cas9 system ( GSC ) for Arabidopsis gene modification in male gametocytes, constructed using a SPOROCYTELESS ( SPL ) genomic expression cassette. Four loci in two endogenous genes were targeted by both systems for comparative analysis. Mutations generated by the GSC system were rare in T1 plants but were abundant (30%) in the T2 generation. The vast majority (70%) of the T2 mutant population generated using the UC system were chimeras while the newly developed GSC system produced only 29% chimeras, with 70% of the T2 mutants being heterozygous. Analysis of two loci in the T2 population showed that the abundance of heritable gene mutations was 37% higher in the GSC system compared to the UC system and the level of polymorphism of the mutations was also dramatically increased with the GSC system. Two additional systems based on germ‐line‐specific promoters ( pDD 45‐ GT and pLAT 52‐ GT ) were also tested, and one of them was capable of generating heritable homozygous T1 mutant plants. Our results suggest that future application of the described GSC system will facilitate the screening for targeted gene modifications, especially lethal mutations in the T2 population.