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.

不同结构的DNA激活序列对Cas12a反式切割活性的影响

CRISPR-Cas12a系统的反式切割活性在其识别特定的DNA激活序列后被激活,这不仅能实现特定DNA靶标的直接定量分析,同时也为构建针对多种生物标志物的体外传感体系带来了新的思路。然而,已有文献中所采用的双链DNA(dsDNA)和单链DNA(ssDNA)激活序列结构多种多样,缺乏全面、系统的设计指导原则。针对该问题,该文系统研究了不同结构的DNA激活序列对LbaCas12a反式切割活性的影响。通过对比研究,得出以下结论:(1)前间区序列邻近基序(PAM)位点有助于LbaCas12a更高效地靶向结合dsDNA激活序列和ssDNA激活序列;(2)PAM近端区域缺少序列片段会降低Cas12a-crRNA定位激活序列的效率;(3)删除PAM远端序列片段有利于增强LbaCas12a的反式切割活性;(4)由于省略了dsDNA解链过程,ssDNA激活序列在激活LbaCas12a的反式切割活性方面普遍比dsDNA激活序列产生的效果更好。根据这些发现,该文提出了一种LbaCas12a所青睐的高效激活序列结构,其激活的LbaCas2a反式酶切活性较采用含PAM位点的标准dsDNA激活序列高出3.7倍。研究结果为构建基于CRISPR-Cas12a的高效体外生物传感系统提供了重要支撑。

Gene editing for corneal disease management

Gene editing has recently emerged as a promising technology to engineer genetic modifications precisely in the genome to achieve long-term relief from corneal disorders.Recent advances in the molecular biology leading to the development of clustered regularly interspaced short palindromic repeats(CRISPRs) and CRISPR-associated systems,zinc finger nucleases and transcription activator like effector nucleases have ushered in a new era for high throughput in vitro and in vivo genome engineering.Genome editing can be successfully used to decipher complex molecular mechanisms underlying disease pathophysiology,develop innovative next generation gene therapy,stem cell-based regenerative therapy,and personalized medicine for corneal and other ocular diseases.In this review we describe latest developments in the field of genome editing,current challenges,and future prospects for the development of personalized genebased medicine for corneal diseases.The gene editing approach is expected to revolutionize current diagnostic and treatment practices for curing blindness.

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.

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.