Recently, academician Liu Yaoguang's team from College of Life Sciences, South China Agricultural University, The State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, and Guangdong Laboratory for Lingnan Modern Agriculture, published a research paper entitled "The ScCas9++ variant expands the CRISPR toolbox for genome editing in plants" online in Journal of Integrative Plant Biology. This research uses optimized CRISPR/ScCas9++ new variants derived from Streptococcus canis to develop a wide-targeted plant genome editing and cytosine single-base editor, providing new opportunities for plant functional genomics research and crop genetic improvement research tool.
At present, CRISPR/Cas genome editing technology has become an important tool for functional genomics research and crop genetic improvement. However, its editing range is often limited by PAM motifs and their editing windows. Therefore, the development of Cas variants with a wider range of PAM recognition is an important research direction to expand the scope of editing use of CRISPR/Cas systems. ScCas9 is a Cas9 protein derived from Streptococcus canis, which has a high similarity of 89.2% with the most commonly used SpCas9 protein (Streptococcus pyogenes, recognizing NGG-PAM) and recognizes NNG-PAM with a wider targeting range, but its editing efficiency in animals and plants is low and difficult to promote for use. Previously, evolutionary-type ScCas9+ and ScCas9++ variants were obtained by replacing amino acids in the PAM-DNA interaction domain and loop motif of ScCas9, and showed high editing activity and a wide editing range in animal cells. However how the editing ability of ScCas9+ and ScCas9++ variants in plants remains unclear.
In order to expand the targeting range of plant CRISPR/Cas systems, academician Liu Yaoguang's team optimized the coding DNA sequences of ScCas9 and its two evolved ScCas9+ and ScCas9++ variants based on rice codon preference and systematically studied their editing efficiency for 22 target sites. The results showed that the editing efficiency of ScCas9+ was higher than that of ScCas9 and ScCas9+, but it mainly recognized NGG-PAM targets, while other NNG targets had very low recognition ability. Furthermore, the researcher fused the evolved cytidine deaminase PmCDA1 with ScCas9n++ to generate a new evoBE4max-type cytidine base editor, termed PevoCDA1-ScCas9n++. This base editor achieved stable and efficient multiplex-site base editing at NNG-PAM sites with wider editing windows (C-1 to C17) and without target sequence context preference. Multiplex-site base editing of the rice genes OsWx (3 targets) and OsEui1 (2 targets) achieved simultaneous editing and produced new rice germplasm.
This study lays a foundation and provides ideas for the further development and application of ScCas9++ variants in plant genome editing. Its notch enzyme variant ScCas9n++ has potential applications in base editor development. This CBE editor PevoCDA1-ScCas9n++ provides a more effective tool for single-base editing of multiple targets, which is expected to be widely used in gene function screening, large-scale saturation mutation, editing regulatory elements, introducing premature stop codons or performing alternative splicing.
Collected by Lifeasible that has developed a transgenic plant platform based on CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein9) to provide engineered plants with enhanced properties such as high yields and nutrients, resistance to disease, pests, or drought tolerance.