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The type I clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system is one of five adaptive immune systems and exists widely in bacteria and archaea. In this study, we showed that  Xanthomonas oryzae  pv.  oryzae  ( Xoo ) possesses a functional CRISPR system by engineering constructs mimicking its CRISPR cassette. CRISPR array analysis showed that the TTC at the 5′-end of the target sequence is a functional protospacer-adjacent motif (PAM) of CRISPR. Guide RNA (gRNA) deletion analysis identified a minimum of 27-bp spacer that was required to ensure successful self-target killing in PXO99 A  strain. Mutants with deletion of individual  Cas  genes were constructed to analyze the effects of Cas proteins on mature CRISPR RNA (crRNA), processing intermediates and DNA interference. Results showed that depleting each of the three genes,  cas5d ,  csd1 , and  csd2  inactivated the pre-crRNA processing, whereas inactivation of  cas3  impaired in processing pre-crRNA. Furthermore, the  Xoo  CRISPR/Cas system was functional in  Pseudomonas syringae  pv.  tomato . Collectively, our results would contribute to the functional study of CRISPR/Cas system of  Xoo , and also provide a new vision on the use of bacterial endogenous systems as a convenient tool for gene editing.

Functional Identification of the Xanthomonas oryzae pv. oryzae Type I-C CRISPR-Cas System and Its Potential in Gene Editing Application