Expanding the targeting scope of CRISPR/Cas9-mediated genome editing by Cas9 variants in Brassica

This study is led by Professor Jun Li (College of Life Sciences, Hebei Agricultural University, Baoding, Hebei, China). The authors used Cas9 variants to expand the targeting of CRISPR/ Cas9-mediated genome editing in Brassica. The authors first performed PAM analysis of the genomes of Chinese cabbage (Brassica rapa spp. pekinensis) and cabbage (Brassica oleracea var. capitata) and found that Cas9-NG/SpG could potentially target at least twice as many sites in the genome than SpCas9. And SpRY could target all sites with NNN PAMs. Subsequently, the author constructed CRISPR/Cas9-NG, CRISPR/SpG and CRISPR/SpRY gene editing systems and transformed them into Chinese cabbage and cabbage protoplasts. The editing efficiency and bias of Cas9-NG, SpG and SpRY variants at different PAM sites were compared. NGS sequencing results showed that both Cas9-NG and SpG were able to recognize a broad range of NGN PAMs in Brassica, and SpG had a higher editing efficiency than Cas9-NG at NGT and NGC PAMs; SpRY is able to target near PAM-less sites and has higher editing efficiency at the NRN PAM site than NYN.

To assess the activity of SpRY in planta, edited regenerated cabbages were obtained, indicating that SpRY was able to perform targeted mutagenesis in stable cabbage plants in a PAM-less fashion. To introduce precise adenine base editing (ABE) at near-PAM-less sites, SpRY(D10A) nickase was fused to TadA8e, resulting in pBSE-SpRYn-ABE8e. PCR/RE assay and Sanger sequencing confirmed that the SpRY-ABE8e achieved efficient A-to-G substitutions at the NNN PAM site in Chinese cabbage.

In summary, this work has developed gene-editing toolboxes for identifying non-canonical PAMs in Brassica, greatly expanding the targeting scope of genome editing by Cas9 variants. It paves the way for future basic research and genetic improvement in Brassica.

See the article:

Expanding the targeting scope of CRISPR/Cas9-mediated genome editing by Cas9 variants in Brassica

https://link.springer.com/article/10.1007/s42994-024-00155-7