PAM-less plant genome editing using a CRISPR–SpRY toolbox

Jan 5, 2021Nature plants

Plant genome editing without PAM restrictions using a CRISPR-SpRY tool

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Abstract

Genome editing using the engineered SpRY Cas9 variant achieved up to 79% editing efficiency at relaxed PAM sites in rice plants.

The SpRY Cas9 variant allows targeted mutagenesis in rice and Dahurian larch at sites previously restricted by PAM requirements.
Highly efficient DNA editing was accomplished through non-homologous end joining in both rice transgenic lines and Dahurian larch protoplasts.
A cytosine base editor was developed that generated new herbicide-resistant alleles in rice.
An adenine base editor based on SpRY-ABE8e demonstrated significant editing capacity at relaxed PAM sites.
Secondary off-target effects were observed due to self-editing of single guide RNAs, indicating a need for improved sgRNA design in using the SpRY toolbox.

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The rapid development of the CRISPR-Cas9, -Cas12a and -Cas12b genome editing systems has greatly fuelled basic and translational plant research. DNA targeting by these Cas nucleases is restricted by their preferred protospacer adjacent motifs (PAMs). The PAM requirement for the most popular Streptococcus pyogenes Cas9 (SpCas9) is NGG (N = A, T, C, G), 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 variant. Highly efficient targeted mutagenesis can be readily achieved by SpRY at relaxed PAM sites in the Dahurian larch protoplasts and in rice transgenic lines through non-homologous end joining (NHEJ). Furthermore, an SpRY-based cytosine base editor was developed 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.) and SpRY-ABE8e was able to target relaxed PAM sites in rice plants, achieving up to 79% editing efficiency with high product 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 secondary 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. 1-6 7 8 9

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PAM-less plant genome editing using a CRISPR–SpRY toolbox

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