Fusing T5 exonuclease with Cas9 and Cas12a increases the frequency and size of deletion at target sites.

CRISPR/Cas systems, especially CRISPR/Cas9, generally result in small insertions/deletions, which are unlikely to eliminate the functions of regulatory and other non-coding sequences. To generate larger genomic deletions usually requires the use of pairs of guide RNAs. Here we show that it is possible to create such deletions with a single guide RNA by fusing Cas9 or Cas12a with T5 exonuclease (T5exo). These fusion constructs were found to increase both the frequency and size of deletions at target loci in rice protoplasts and seedlings. Moreover, the genome editing efficiencies of Cas9 and Cas12a were also enhanced by fusion with T5 exonuclease. These T5exo-Cas fusions expand the CRISPR toolbox, and facilitate knockout of regulatory and non-coding DNA sequences. From a wider standpoint, our results suggest a general strategy for producing larger deletions using other Cas nucleases.