Report: PAM-less plant genome editing using a CRISPR–SpRY toolbox
Host: Wu Yuechao
Date: 08 Sep 2021
The rapid development of the CRISPR–Cas9, –Cas12a and – Cas12b genome editing systems has greatly fuelled basic and translational plant research1–6. DNA targeting by these Cas nucleases is restricted by their preferred protospacer adja- cent motifs (PAMs). The PAM requirement for the most popu- lar Streptococcus pyogenes Cas9 (SpCas9) is NGG (N = A, T, C, G)7, limiting its targeting scope to GC-rich regions. Here, we demonstrate genome editing at relaxed PAM sites in rice (a monocot) and the Dahurian larch (a coniferous tree), using an engineered SpRY Cas9 variant8. Highly efficient targeted mutagenesis can be readily achieved by SpRY at relaxed PAM sites in the Dahurian larch protoplasts and in rice trans- genic lines through non-homologous end joining (NHEJ). Furthermore, an SpRY-based cytosine base editor was devel- oped and demonstrated by directed evolution of new herbicide resistant OsALS alleles in rice. Similarly, a highly active SpRY adenine base editor was developed based on ABE8e (ref. 9) and SpRY-ABE8e was able to target relaxed PAM sites in rice plants, achieving up to 79% editing efficiency with high prod- uct purity. Thus, the SpRY toolbox breaks a PAM restriction barrier in plant genome engineering by enabling DNA editing in a PAM-less fashion. Evidence was also provided for second- ary off-target effects by de novo generated single guide RNAs (sgRNAs) due to SpRY-mediated transfer DNA self-editing, which calls for more sophisticated programmes for designing highly specific sgRNAs when implementing the SpRY genome editing toolbox.