2.2 Optimizing gRNA expression

CRISPR / Cas9's simple and powerful multi-site editing capability is one of its significant advantages. Realizing multi-site editing requires simultaneous expression of multiple gRNA molecules. At present, there are mainly three strategies to use multiple vectors to simultaneously express multiple gRNAs. The first is to clone multiple expression units in a plasmid vector, each unit containing a Pol III promoter and gRNA. The second is to use Csy4 nuclease to cleave transcripts containing multiple gRNAs into a single gRNA, where the gRNAs are linked by Csy4's RNA substrate. Third, the endogenous transfer RNA (tRNA) of the organism is fused with gRNA, and multiple tRNA-gRNA structures are arranged in series into one polycistronic. The tRNA processing enzyme in the organism is used to convert this polycistronic. The daughter cuts into mature gRNA for multi-site editing. In addition, self-cleaving ribozyme can also be used for the expression of multiple gRNAs. These different methods provide mature multi-site CRISPR / Cas9 genome editing technologies for plant genetic manipulation.

2.3 Limitations of Reducing PAM

DNA target sites must be present PAM is the main factor limiting CRISPR / Cas9 for genome editing. According to the sequence analysis of the reference genomes of several model plants, the average genome has an average of 6−11 PAM (5′-NGG-3 ′) per 100 bp. Although this frequency has basically met the needs of conventional gene function research, it has greatly limited the scope of CRISPR / Cas9 applications.

In order to eliminate or reduce the PAM-dependent restriction of CRISPR / Cas9, it is necessary to find or create CRISPR / Cas9 that recognizes different PAM sequences for genome editing. In the past years, several CRISPR / Cas9 systems have been discovered, greatly expanding the space for CRISPR / Cas targeted gene editing. In addition, the researchers modified Cas9, the most commonly used Streptococcus pyogenes, to modify the amino acid sites related to PAM recognition in Cas9 protein, and obtained recognition 5′-NGAN-3 ′ and 5′-NGNG-3 ′. Cas9 mutants with different PAM sequences and have been used for plant genome editing. To synthesize these new developments, the existing CRISPR / Cas9 tools basically have the ability to target the whole genome.

3.Beyond CRISPR / Cas9

3.1 Targeted single base editing

In genome editing technology, the most common application is to introduce indel at the target site through CRISPR / Cas9, thereby destroying the function of the target gene. However, in the practice of genetic improvement of crops, it is difficult to obtain materials of breeding or application value by completely destroying the function of the gene. More often, it is necessary to modify the function of the gene by directional modification of a single or several bases, so as to improve the crop Agronomic traits. Therefore, efficient and targeted transformation of the genome sequence is the key to CRISPR / Cas9 technology for genetic improvement practices, and single base editing technology is meeting this need.



Figure. The principle of targeted editing of a single base based on CRISPR / Cas9

The principle of targeted editing of a single base based on CRISPR / Cas9 is shown in Figure above. Cas9n (or dCas9) is fused with Cytidine deaminase. When gRNA guides the fusion protein to the target site, the target site Nearby cytosine C is converted to uracil U by deaminase, and U has the same base pairing rules as thymine T. U will be converted to T when DNA is copied or repaired, thus completing the C → T base Conversion. In order to improve the efficiency of C → U → T, related components will be added to the editing tool to suppress the recovery of U → C. At present, there are two successful single-base editing tools: one is the BE (Baseeditor) system constructed by David Liu's laboratory at the Massachusetts Institute of Technology, which is obtained by fusing dCas9 or Cas9n with mammalian deaminase APOBEC1. The second is the Target-AID, CRISPR-X and TAM systems obtained by fusing dCas9 or Cas9n with AID (Activation-induced deaminase). These different single base editing tools are slightly different in specific design, and the window of target bases that can be edited are also slightly different, but high efficiency C → T single base replacement can be achieved in animal cells.

Although Cas9-mediated knock-in can also be used to achieve single-base substitution, the efficiency and ease of implementation of HDR are incomparable with single-base editing tools such as BE and Target-AID / TAM / CRISPR-X. When editing a single base on both APOE4 and p53 genes in a mammalian cell line, Cas9-mediated HDR has a base conversion efficiency of up to 0.3%, while the efficiency of the BE3 system reaches a surprising 58% −75 % (APOE4) and 3% −8% (p53). The latest results also indicate that the single-base editing tools engineered with CRISPR / Cas9 are very specific and no off-target editing was detected. These CRISPR single-base editing tools have taken gene editing technology to a new level, laying an important foundation for CRISPR / Cas9 technology for clinical treatment, basic research and crop genetic improvement.

Because single-base editing has great application potential in crop genetics and breeding, these editing techniques have been rapidly applied to crops. For example, if BE (Cas9n-APOBEC1) is used for rice, wheat, and corn, the exact replacement of C → T can be realized in the 3-9 bp edit window of protospacer, with a maximum efficiency of 43.48%. Target-AID was used in rice, and single-base substitution of the ALS (Acetolactate Synthase) gene yielded herbicide-resistant rice material. These successful examples initially demonstrate the great potential of CRISPR / Cas9 single base editing.

3.2 Genome editing technology based on CRISPR / Cpf1 system

The huge success of CRISPR / Cas9 from S. pyogenes for genome editing has prompted researchers to find and modify other CRISPR / Cas9 systems to expand the gene editing toolbox. The most potential currently is the CRISPR / Cpf1 system, which is considered to be a new generation of the best in genome editing tools. In 2015, the Zhang Feng laboratory at the Massachusetts Institute of Technology in the United States first discovered the Cpf1 endonuclease, a member of the 5th subfamily of the class II CRISPR system. The three highly homologous proteins AsCpf1, LbCpf1 and FnCpf1 of the genus Franisella novicida have RNA-guided endonuclease activity, and the efficiency of AsCpf1 and LbCpf1 for animal genome editing is close to the original CRISPR / Cas9 system.

To be continued in Part Four…