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Backgorund  Environmental stresses including abiotic stresses and biotic stresses limit yield of plants. Stress-tolerant breeding is an efficient way to improve plant yield under stress conditions. Genome editing by CRISPR/Cas9 can be used in molecular breeding to improve agronomic traits in crops, but in most cases, with fitness costs. The plant hormone ABA regulates plant responses to abiotic stresses via signaling transduction. We previously identified AITRs as a family of novel transcription factors that play a role in regulating plant responses to ABA and abiotic stresses. We found that abiotic stress tolerance was increased in the single, double and triple  aitr  mutants. However, it is unclear if the increased abiotic stress tolerance in the mutants may have fitness costs.    Results  We report here the characterization of  AITRs  as suitable candidate genes for CRISPR/Cas9 editing to improve plant stress tolerance. By using CRISPR/Cas9 to target  AITR3  and  AITR4  simultaneously in the  aitr256  triple and  aitr1256  quadruple mutants respectively, we generated Cas9-free  aitr23456  quintuple and  aitr123456  sextuple mutants. We found that reduced sensitivities to ABA and enhanced tolerance to drought and salt were observed in these mutants. Most importantly, plant growth and development was not affected even in the  aitr123456  sextuple mutants, in whom the entire  AITR  family genes have been knocked out, and the  aitr123456  sextuple mutants also showed a wild type response to the pathogen infection.    Conclusions  Our results suggest that knockout of the  AITR  family genes in Arabidopsis enhanced abiotic stress tolerance without fitness costs. Considering that knock-out a few  AITRs  will lead to enhanced abiotic stress tolerance, that AITRs are widely distributed in angiosperms with multiple encoding genes,  AITRs  may be targeted for molecular breeding to improve abiotic stress tolerance in plants including crops.

Knockout of the entire family of AITR genes in Arabidopsis leads to enhanced drought and salinity tolerance without fitness costs