Rapid and efficient CRISPR/Cas9-mediated genome editing in potato via hairy root induction

Potato is one of the most important crops, and its trait improvement through genetic engineering is of utmost need. CRISPR/Cas9-mediated genome editing tool and its advancement revolutionized genetic and metabolic engineering. Cas9 endonuclease requires gRNA and PAM for targeted genome editing. Delivery of CRISPR/Cas9 component via Rhizobium is most commonly used. Different factors affecting genetic transformation, for example bacterial strains, bacterial density, co-cultivation duration, acetosyringone, explants type, and cultivars, were optimized for Indian potato. Wild-type Rhizobium rhizogenes strain A4 showed the highest (60.7 ± 6.9%) hairy root induction followed by ATCC15834 (53.4 ± 10.7%). The highest transformation efficiency (68.8 ± 6.8%) was achieved onto Murashige and Skoog medium (MS) supplemented with 0.5 mg L−1 naphthaleneacetic acid (NAA). The putative transgenic hairy roots were induced within 8 to 10 d on hygromycin-containing media. A4 strain carrying CRISPR/Cas9 vector was used to target the StbHLH47. A total of 32 possible target sites near to PAM region were identified in the 500-bp sequence of StbHLH47. The target site adjacent to the ATG start codon was selected, and the construct was prepared into the pHSE401 vector. PCR amplification of rolB, HptII, and Cas9 gene–specific primers showed the integration of CRISPR/Cas9 components in the potato genome. Transgenic hairy root lines showed 2 to 7 nucleotide deletion at the target site of StbHLH. The editing frequency was calculated to be 15.3% for StbHLH47. This study reports a fast and efficient protocol for hairy root induction by Rhizobium rhizogenes to analyze editing in the potato genome within 18 to 20 d. The developed protocol will be very useful for studying gene function using CRISPR in the area of root biology.