Gene

Cell biology

Molecular biology

Bacteria

Biology

Genome editing

Trans-activating crRNA

CRISPR

Xanthomonas

Xanthomonas citri

Computational biology

Single-nucleotide polymorphism

Genetics

Genome

Genotype

Citrus canker

Effector

Indel

Gene expression

Agroinfiltration

Plant Biotechnology Journal

Summary Recently, CRISPR ‐Cas12a (Cpf1) from Prevotella and Francisella was engineered to modify plant genomes. In this report, we employed CRISPR ‐LbCas12a (LbCpf1), which is derived from Lachnospiraceae bacterium ND 2006 , to edit a citrus genome for the first time. First, LbCas12a was used to modify the Cs PDS gene successfully in Duncan grapefruit via Xcc‐facilitated agroinfiltration. Next, LbCas12a driven by either the 35S or Yao promoter was used to edit the PthA4 effector binding elements in the promoter ( EBE P thA4 ‐Cs LOBP ) of Cs LOB 1 . A single cr RNA was selected to target a conserved region of both Type I and Type II Cs LOBP s, since the protospacer adjacent motif of LbCas12a ( TTTV ) allows cr RNA to act on the conserved region of these two types of Cs LOBP . Cs LOB 1 is the canker susceptibility gene, and it is induced by the corresponding pathogenicity factor PthA4 in Xanthomonas citri by binding to EBE P thA4 ‐Cs LOBP . A total of seven 35S‐LbCas12a‐transformed Duncan plants were generated, and they were designated as #D 35 s1 to #D 35 s7, and ten Yao‐LbCas12a‐transformed Duncan plants were created and designated as #D yao 1 to #D yao 10. LbCas12a‐directed EBE P thA4 ‐Cs LOBP modifications were observed in three 35S‐LbCas12a‐transformed Duncan plants (#D 35 s1, #D 35 s4 and #D 35 s7). However, no LbCas12a‐mediated indels were observed in the Yao‐LbCas12a‐transformed plants. Notably, transgenic line #D 35 s4, which contains the highest mutation rate, alleviates XccΔpthA4: dCsLOB 1.4 infection. Finally, no potential off‐targets were observed. Therefore, CRISPR ‐LbCas12a can readily be used as a powerful tool for citrus genome editing.

<scp>CRISPR</scp>‐LbCas12a‐mediated modification of citrus

CRISPR‐LbCas12a‐mediated modification of citrus