Iwen, T. | Search bioRxiv

bioRxiv2023

Efficient scar-free knock-ins of several kilobases by engineered CRISPR/Cas endonucleases

In plants and mammals, non-homologous end-joining is the dominant pathway to repair DNA double strand breaks, making it challenging to generate knock-in events. Using a transient assay in Nicotiana benthamiana we identified two groups of exonucleases, respectively from the Herpes Virus and from the bacteriophage T7 families, hat confer up to 38-fold increase in HDR frequencies when fused to Cas9. We achieved precise and scar-free insertion of several kilobases of DNA both in transient and stable transformation systems. In Arabidopsis thaliana, fusion of Cas9 to a Herpes Virus family exonuclease leads to 10-fold higher frequencies of knock-ins. Our results open perspectives for the routine production of knock-in and gene replacement events in plants. One-Sentence SummaryFusions of CRISPR endonucleases to specific 5'-exonucleases leads to significant increase in scar-free multikilobase knock-ins.

molecular biology↗

bioRxiv2022

DNA double strand breaks lead to de novo transcription and translation of damage-induced long RNAs in planta

DNA double strand breaks (DSBs) are lethal threats that need to be repaired. Although many of the proteins involved in the early steps of DSB repair have been characterized, recent reports indicate that damage induced long and small RNAs also play an important role in DSB repair. Here, using a Nicotiana benthamiana transgenic line originally designed as a reporter for targeted knock-ins, we show that DSBs generated by Cas9 induce the transcription of long stable RNAs (damage-induced long RNAs - dilRNAs) that are translated into proteins. Using an array of single guide RNAs we show that the initiation of transcription takes place in the vicinity of the DSB. Single strand DNA nicks are not able to induce transcription, showing that cis DNA damage-induced transcription is specific for DSBs. Our results support a model in which a default and early event in the processing of DSBs is transcription into RNA which, depending on the genomic and genic context, can undergo distinct fates, including translation into protein, degradation or production of small RNAs. Our results have general implications for understanding the role of transcription in the repair of DSBs and, reciprocally, reveal DSBs as yet another way to regulate gene expression.

molecular biology↗