Development And Application Of CRISPR-Cpf1p And CRISPR-xCas9p Genome Editing System In Plants

Clustered regularly interspaced short palindromic repeats(CRISPR)gene editing system is a new generation of genome editing technology,which has the advantages of simple operation and high efficiency,and is widely used in plant gene function research and genetic improvement.At present,the most widely used CRISPR-associated endonuclease 9(Cas9)gene editing system is restricted by the precursor adjacent motif(PAM)NGG.To expand the flexibility of target selection and reduce the possibility of occasional off-target cases,CRISPR-associated endonuclease 12a(Cas12a)(also known as Cpf1)and CRISPR-x Cas9 variant system have been established.In this study,we developed a plant CRISPR Cpf1p and x Cas9p gene editing system,and successfully knocked out 6 rice negative regulatory resistance genes,creating disease resistance rice materials.The main results are as follows:1.We optimized the codon of Francisella novicida U112 Cpf1(Fn Cpf1)and Lachnospiraceae bacterium ND2006 Cpf1(Lb Cpf1)for plant gene preference to generate Fn Cpf1p and Lb Cpf1p.These genes were linked to the p Ubi promoter and cloned into the p CAMBIA1300 binary vector to form a p YLCRISPR/Cpf1p binary vector.2.A series of cr RNA vectors preparation of the expression cassette of cr RNA(CRISPR-derived RNA)were constructed.Using Golden Gate or Gibson assembly method,the amplified cr RNA expression cassettes can be integrated into the p YLCRISPR/Cpf1p binary vectors.3.Three Fn Cpf1p/cr RNA and three Lb Cpf1p/cr RNA gene editing vectors for two targeting sites were constructed using the above vector system.With these vecters,four negative regulatory disease-resistant genes Os Pi21,Os ELF3-2,Os Pti1a and Os GA20ox3were knocked out and 121 positive plants were obtained.Target efficiency and editing characteristics of each vector were analyzed.Of 68 analyzed targeting events by the Fn Cpf1p system,the average mutation rate was 18.5%.Of 53 analyzed target sites targeting events by Lb Cpf1p/cr RNA system,the average mutation rate was 20.4%.Their cutting sites were at 15-23 bp downstream positions to PAM site,and most of the mutations were deletion of 4-8 bp.Two Fn Cpf1p 6-target editing vectors were constructed,and it was found that a single U6 promoter could drive multiple tandem cr RNA to knockout genes.4.The xCas9p gene was assembled by overlapping PCR and linked with the p Ubi promoter to form a series of p YLCRISPR/x Cas9p binary vectors.5.The structure of sg RNA(single-guide RNA)was optimized and named as sg RNAm(single-guide RNA modification).Using Golden Gate or Gibson Assembly method,the amplified sg RNAm expression cassettes were integrated into a binary vector containing the x Cas9 expression cassette to form a x Cas9p/sg RNAm vector system.6.Three xCas9p/sg RNAm double-target vectors for knockout of three rice negative regulatory disease-resistant genes,Os HDT701,Os ELF3-2 and Os Bsr-k1 were constructed and one cas9p/sg RNA double-target vector and one cas9p/sg RNAm double-target vector were constructed as control.Totally,122 positive knockout plants were obtained.The results show that sg RNAm can effectively improve the efficiency of gene editing.When PAM is NGG,the editing efficiency of x Cas9p system is equivalent to that of Cas9p system,but when PAM is NGA,the efficiency of x Cas9p system reduce to only 7.1%,and when PAM is NGT and NGC,x Cas9p system fails in editing.7.By analyzing several resultant Os Pi21-KO and Os HDT701-KO lines were obtained by using the above-mentioned gene editing system.T-DNA free knockout lines with steadily inherited mutation sites were isolated in T₁ generation of transgenic rice Nine putative off-target sites were analyzed,The results showed that all sites were normal.8.The disease resistance and the main agronomic characters of three T-DNA free Os Pi21-KO and Os HDT701-KO lines were investigated.The results showed that compared with wildtype plants,Os Pi21-KO lines showed strong blast resistance;the Os HDT701-KO lines has strong resistance to rice blast and bacterial blight.The main agronomic traits of Os Pi21-KO lines did not change,but the tiller number and seed setting rate were reduced in Os HDT701-KO lines.In this study,the Cpf1p and xCas9p gene editing systems were developed in plants,which effectively expanded the flexibility of target selection in Cas9p system,and successfully created disease-resistant rices,providing new technical approaches for crop improvement by gene editing.