AUTHOR=Liu Qibing , Wang Siwei , Long Juying , Chen Zhuoyue , Yang Bing , Lin Fei 

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

JOURNAL=Frontiers in Microbiology

VOLUME=12

YEAR=2021

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2021.686715

DOI=10.3389/fmicb.2021.686715

ISSN=1664-302X

ABSTRACT=<p>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 <italic>Xanthomonas oryzae</italic> pv. <italic>oryzae</italic> (<italic>Xoo</italic>) 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<sup>A</sup> strain. Mutants with deletion of individual <italic>Cas</italic> 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, <italic>cas5d</italic>, <italic>csd1</italic>, and <italic>csd2</italic> inactivated the pre-crRNA processing, whereas inactivation of <italic>cas3</italic> impaired in processing pre-crRNA. Furthermore, the <italic>Xoo</italic> CRISPR/Cas system was functional in <italic>Pseudomonas syringae</italic> pv. <italic>tomato</italic>. Collectively, our results would contribute to the functional study of CRISPR/Cas system of <italic>Xoo</italic>, and also provide a new vision on the use of bacterial endogenous systems as a convenient tool for gene editing.</p>