2) Targeted knock-in. Knock-in a sequence on a plant's chromosome has always been a difficult technical problem, and TALEN and CRISPR / Cas9 technologies have greatly reduced the difficulty of this technology. Knock-in relies on DSB's Homology-directed repair (HDR) repair pathway (Figure A): After Cas9 / gRNA cuts the target site, if the cell has DNA homologous to the target site sequence in the case of a donor (DNAdonor) fragment, the gene fragment located on the donor DNA is integrated into the DSB location by HDR recombination. Although CRISPR / Cas9 technology can be used in plant protoplasts to achieve Knock-in or targeted gene replacement, it is difficult to succeed in stable plants.
Currently, there are two more general strategies that can be used to improve the efficiency of plant knock-in. The first is to use wheat dwarf virus (WDV) as a vector for DNA donors. By increasing the number of copies of donor DNA, the efficiency of Knock-in is greatly improved. This strategy has been validated in two major crops, rice and wheat, and Knock-in has a maximum efficiency of 19.4% in rice. The second is to use Cas9 and a pair of gRNAs to “cut” a sequence from a DNA donor and then “paste” it to a target site in the plant genome. This strategy utilizes the NHEJ pathway that dominates DSB repair. In 2016, a laboratory of the Institute of Genetics and Developmental Biology of the Academy of Sciences collaborated to achieve this "cut / paste" target in rice using clever target site design using CRISPR / Cas9 Knock in and replace genes. Based on CRISPR / Cas9, these new strategies make it possible for knock-in to be used efficiently in plant genetic modification.
1.2.2 Targeted gene transcription regulation
In addition to the genome editing function, the high scalability of CRISPR technology ensures that this technology can be fused with multiple functional proteins to perform other genetic operations. The core of this technology is the ability of gRNAs to target specific sites on the genome, using dCas9 (Dead Cas9, Cas9 without DNA-cleaving activity) and gRNAs to transport effector proteins of different functions to specific locations on the chromosome to function (Figure C). For example, dCas9 can be fused to an activation domain (AD), and gRNA can be used to guide dCas9-AD to the promoter region to achieve transcriptional activation of target genes. In this technique, multiple gRNAs are often required to simultaneously target the promoter of the target gene in order to effectively increase the expression of the target gene. And another elaborate strategy addresses this problem: using gRNAscaffold as a platform for recruiting transcriptional regulatory components to regulate target gene expression. For example, MS2 (RNA ligand) is fused to gRNA and MS2's specific binding protein MCP is fused to the AD fragment, so that the dCas9 / gRNA-MS2 / MCP-AD complex is targeted to activate transcription on the gene promoter of interest. The ingenuity of this technology is that multiple MS2s can be integrated into the gRNA scaffold sequence so that a single gRNA can be used to recruit multiple MCP-ADs to effectively activate target gene expression. Similar to CRISPR / Cas9-based targeted gene transcription activation, using different functional proteins (transcriptional suppression, epigenetic modified protein elements) on the dCas9 / gRNA platform can achieve different transcriptional suppression or epigenetic regulation. These genome-editing-derived technologies provide richer and more convenient genetic tools for biological research.
Figure. Major applications of the CRISPR / Cas9 system in plants
2.Optimization of CRISPR / Cas9 technology
2.1 Reduce CRISPR / Cas9 off-target
Off-target has been a major problem for CRISPR / Cas9 technology. When the CRISPR / Cas9 genome editing technology was born, it was reported that Cas9 / gRNA had an off-target effect in animal cell lines: some DNA sites (off-target sites) that did not exactly match the gRNA guide sequence were also off-target edited by Cas9 and introduced unexpected genetic mutation (Off-target effects). The existence of off-target effects has become the biggest deficiency of CRISPR / Cas9. To solve this problem, scientists have made many positive attempts. In earlier studies, the Cas9 point mutation (D10A or H804A) was modified into a nicking enzyme (Cas9nickase, Cas9n) or dCas9 and FokI were fused into a nicking enzyme. This required the design of 1 pair of gRNAs to target 1 site. The formation of a DSB reduces the probability of missed targets by several orders of magnitude. Other studies have shown that shortening the guide sequence of gRNA to 17-18 nt can reduce the risk of off-target; or the fusion of Cas9 protein with other DNA-binding domains can also effectively reduce the off-target rate. Although these strategies greatly reduce off-target effects, they also complicate the technology and do not substantially improve the specificity of Cas9. As the structure of the Cas9 / gRNA complex is resolved, researchers have designed Cas9 variants with high cleavage activity and specificity. These "high-fidelity" versions of Cas9 have zero tolerance for base mismatches between DNA and gRNA. Yes, it can be used to achieve precise genetic manipulation, which basically solves the problem of CRISPR / Cas9 off-target.
In plant CRISPR / Cas9 gene editing experiments, the off-target phenomenon was not as severe as in animals, but individual off-target editing was also found, which may be caused by the absence of highly specific gRNA in the design of target sites. The results of genome-wide sequence analysis show that in species with small genomes and low sequence repetitiveness, such as Arabidopsis thaliana and rice, it is possible to design enough high specific gRNAs to edit 90% of genes. There are many bioinformatics tools available for off-target analysis and design of gRNAs with low off-target probability, such as CRISPR-PLANT, E-CRISP, CRISPR-P, etc. By designing highly specific gRNAs or using a new "high-fidelity" version of Cas9, it is expected to effectively eliminate the impact of CRISPR / Cas9 off-target on plant genome editing.
To be continued in Part Three…