Advances in plant CRISPR genome editing technology (Part Four)

Posted by beauty33 on November 30th, 2019

Compared with Cas9, Cpf1 has the following advantages in genome editing: 1) the guide RNA of Cpf1 is shorter, but the guide sequence (that is, the DNA target site) is longer (~ 24 nt); 2) the specificity of Cpf1 is better than Cas9, Cpf1's off-target phenomenon was not detected in off-target analysis experiments; 3) Cpf1 can cut CRISPR RNA array into multiple guide RNAs, making it easier to implement multi-site editing; 4) Cpf1 recognizes PAM as 5′-TTTN- 3 ′ (AsCpf1 and LbCpf1) and 5′-TTN-3 ′ (FnCpf1), greatly expanding the scope of genome editing; 5) Cpf1 forms a 5 base overhang in the protospacer region of PAM, The type of mutation formed is different from Cas9, and it is easier to introduce indel, which improves the efficiency of knock-out.

These features of Cpf1 give it an advantage in genome editing. Therefore, the CRISPR / Cpf1-based gene editing system is rapidly being used for genome editing and targeted gene transcription regulation in model organisms such as rice and Arabidopsis. The efficiency of using CRISPR / Cpf1 system to target gene knockout in rice is not significantly different from the original CRISPR / Cas9 technology. The CRISPR / Cpf1 system's easy multi-gene editing capabilities have also been validated in rice. In addition, CRISPR / Cpf1 can also be transformed into a tool for targeted gene regulation, achieving targeted gene expression suppression in Arabidopsis. In general, CRISPR / Cpf1 will be a plant genome editing platform comparable or even better than CRISPR / Cas9.

4.Genome editing for crop genetic improvement

Although the CRISPR / Cas9 genome editing technology has been a short-term tool of choice for plant genetic manipulation, it has shown great application value in the genetic improvement of crops. For example, in wheat, genomic editing technology was used to precisely target three copies of the mutated MLO gene, and wheat materials with broad-spectrum resistance to powdery mildew were directly obtained. In rice, the CRISPR / Cas9 system was used in temperature-sensitive nuclear males. Specific mutations were introduced into the sterility gene TMS5, and new sterility line materials were developed, which are expected to accelerate the application of temperature-sensitive nuclear male sterility lines in rice cross breeding. CRISPR / Cas9 has also been transformed into a new tool for resistance to viruses by botanists, using Cas9 and gemini-targeting gRNAs in Arabidopsis and tobacco to achieve plant immunity to target viruses. With the rapid expansion of CRISPR / Cas9 technology, more and more gene-edited crops will enter the practical application stage.

The establishment and maturation of DNA-free genome editing technology will further promote the pace of CRISPR / Cas9 technology for genetic improvement of crops. DNA-free genome editing refers to directly transferring Cas9 protein (or Cpf1) and synthetic gRNA expressed in vitro into plant cells to complete the editing of target genes, and then regenerating the edited cells into complete plants. Since the entire process does not involve any foreign DNA, no additional genetic modification will be produced beyond the edited target site. This technique was first validated in protoplasts of Arabidopsis, tobacco, lettuce, and rice 4 plants. Subsequently, DNA-free CRISPR / Cas9 gene editing technology was established in key food crops such as corn and wheat. DNA-free technology may further eliminate public concerns about genome-edited crops, and is expected to accelerate the application of CRISPR / Cas9 technology in crop genetic improvement.

The emergence of genome-edited crops has also prompted regulators to re-examine crop varieties improved by technologies such as CRISPR / Cas9 and TALEN, especially their differences from traditional genetically modified crops. The USDA has clearly stated that genome-edited improved mushroom and potato varieties can be equivalent to traditional breeding crop varieties because there is no foreign DNA in the final product. With the rapid development of CRISPR / Cas9 technology and its widespread use in different crops, how to manage new crop varieties improved by genome editing technology will be an important issue.

5. Outlook

The emergence of CRISPR / Cas9 technology provides a simple, cheap and accurate genetic manipulation platform, which can not only provide strong support for basic research, but also accelerate the transformation of functional genomics research results into products, injecting new impetus for genetic improvement of crops.