Development and Application of CRISPR/Cas System in Rice

In the past several years, the CRISPR(clustered regularly interspaced short palindromic repeats)/Cas(CRISPR-associated protein) system has been harnessed as an efficient and powerful tool for targeted genome editing in different prokaryotic and eukaryotic species. Here, we review the development and application of CRISPR/Cas system in rice, emphasizing different varieties of CRISPR/Cas systems have been applied, strategies for multiplex editing, methods for precise gene insertion and replacement, and efficient systems for base editing and site-specific transcriptional regulation. In addition, the biosecurity of CRISPR/Cas system is also discussed, including transgene-free methods and off-target effects of CRISPR/Cas system. Thus, the development and application of CRISPR/Cas system will have significant impact on functional genomic research and variety improvement in rice.

Review: Plant Genome Editing Targeted by RNA-Guided DNA Endonuclease CRISPR/Cas9

This review chronicles the development of the research on CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR associated protein 9) during the last 30 years from the discovery of CRISPR sequence, of biological significance and of the molecular mechanism for adaptive bacterial immunity. It describes recent works on structural and functional diversity of CRISPR/Cas systems, and on three-dimensional structure-based improvements of on-target specificity of CRISPR/Cas9 and Cpf1 endonucleases. The review ends with the application of CRISPR/Cas9 to targeted editing of plant genomes. Importantly, plant commodities modified by CRISPR-Cas9 have not been considered as genetically modified organisms (GMO) as long as foreign DNAs from plant pests were not introduced, according to the recent determination by the USDA.

Expanding the range of CRISPR/Cas9-directed genome editing in soybean

The CRISPR/Cas9 system has been widely applied for plant genome editing.The commonly used SpCas9 has been shown to rely on the protospacer adjacent motif(PAM)sequences in the canonical form NGG and non-canonical NAG.Although these PAM sequences are extensively distributed across plant genomes,a broader scope of PAM sequence is required to expand the range of genome editing.Here we report the adoption of three variant enzymes,xCas9,SpCas9-NG and XNG-Cas9,to produce targeted mutation in soybean.Sequencing results determined that xCas9 with the NGG and KGA(contains TGA and GGA)PAMs successfully induces genome editing in soybean genome.SpCas9-NG could recognize NGD(contains NGG,NGA and NGT),RGC(contains AGC and GGC),GAA and GAT PAM sites.In addition,XNG-Cas9 was observed to cleave soybean genomic regions with NGG,GAA and AGY(contains AGC and AGT)PAM.Moreover,off-target analyses on soybean editing events induced by SpCas9 and xCas9 indicated that two high-fidelity Cas9 variants including eSpCas9(enhanced specificity SpCas9)and exCas9(enhanced specificity xCas9)could improve the specificity of the GGA PAM sequence without reducing on-target editing ficiency.These findings significantly expand the scope of Cas9-mediated genome editing in soybean.

Can SpRY recognize any PAM in human cells?

The application of clustered regularly interspaced short palindromic repeats(CRISPR)and CRISPR-associated proteins(Cas)can be limited due to a lack of compatible protospacer adjacent motif(PAM)sequences in the DNA regions of interest.Recently,SpRY,a variant of Streptococcus pyogenes Cas9(SpCas9),was reported,which nearly completely fulfils the PAM requirement.Meanwhile,PAMs for Sp RY have not been well addressed.In our previous study,we developed the PAM Definition by Observable Sequence Excision(PAM-DOSE)and green fluorescent protein(GFP)-reporter systems to study PAMs in human cells.Herein,we endeavored to identify the PAMs of SpRY with these two methods.The results indicated that 5’-NRN-3’,5’-NTA-3’,and 5’-NCK-3’could be considered as canonical PAMs.5’-NCA-3’and 5’-NTK-3’may serve as non-priority PAMs.At the same time,PAM of 5’-NYC-3’is not recommended for human cells.These findings provide further insights into the application of SpRY for human genome editing.