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2019/11/18 ABRC Seminar

Speaker: Dr. Masaki Endo (Senior Researcher, Plant Genetic Engineering Research Unit, Institute of Agrobiological Sciences, National Agriculture and Food Research Organization(NARO), Japan)
Topic: Precision genome editing in plants using natural and engineered CRISPR variants in plants

The Cas9 nuclease from Streptococcus pyogenes (Sp) is commonly used for genome engineering. We previously reported that Cas9 from Staphylococcus aureus (Sa) can be used for targeted mutagenesis in rice and tobacco. SaCas9 has higher sequence recognition capacity than does SpCas9 and is useful for reducing off-target mutations in crops. The protein size of SaCas9 is smaller than that of SpCas9. This property lowers the size of the gene cassette required to express SaCas9. Furthermore, we have shown that SaCas9 can be split, and that split-SaCas9 can induce targeted mutagenesis in Nicotiana benthamiana. We are currently applying split-SaCas9 for virus vector-mediated targeted mutagenesis using two kinds of virus vector systems in tobacco.
Cpf1(Cas12a) is a newly characterized RNA-guided endonuclease that has two distinct features as compared to Cas9. Cpf1 utilizes a thymidine-rich PAM(protospacer adjacent motif) while Cas9 prefers a guanidine-rich PAM. Cpf1 generates DNA ends with a 5’ overhang, whereas Cas9 creates blunt DNA ends after cleavage. Sticky DNA ends could increase the efficiency of insertion and substitution of desired DNA fragments into the Cpf1-cleaved site using complementary DNA ends. Francisella novicida (FnCpf1) can be applied to targeted mutagenesis in rice and tobacco. FnCpf1 can also be used for targeted insertion of a short DNA fragment at a specific site using a PEG-mediated protoplast transformation system.
CRISPR/Cas9-mediated genome editing systems require an appropriate PAM sequence at the target site. SpCas9 recognizes the shortest known PAM, NGG, but we show here that an engineered SpCas9 (SpCas9-NG), recognizing only NG as the PAM sequence, can efficiently mutagenize the rice and Arabidopsis genomes. A SpCas9-NG nickase fused to cytidine deaminase enabled C to T substitutions near the 5’ end of the 20-nt target sequence. Furthermore, SpCas9-NG fused to adenosine deaminase enables A to G substitutions at a position -16 to -13 upstream from the PAM.
Desirable substitutions are not always introduced into a target gene by these base editing systems. Gene targeting (GT) enables one to modify targeted sequences as expected via homologous recombination (HR) using a donor DNA as a template. In rice, precise modification of the targeted sequence via GT using a positive-negative selection system and subsequent removal of the positive selection marker has been developed. We compare base editing systems and a GT system and discuss about future trends of precision genome editing in rice and other plants.

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