Editing efficiencies with Cas9 orthologs, Cas12a endonucleases, and optimal temperature

The advent of CRISPR/Cas technology has made it the genome editing tool of choice in almost all kingdoms of life, including plants, which can have large, highly duplicated genomes. Therefore, finding adequate target sequences that meet the specificities of a given Cas nuclease on any gene of interest remains challenging in many cases. To assess target site flexibility, we tested five different Cas9/Cas12a endonucleases (SpCas9, SaCas9, St1Cas9, Mb3Cas12a, and AsCas12a) in embryogenic rice calli from Taipei 309 at 37 C (optimal temperature for most Cas9/Cas12a proteins) and 27 C (optimal temperature for tissue culture) to assess rate of editing under regular tissue culture conditions through Illumina sequencing.