Rice artificial genome editing system with expanded editing scope and applications thereof

A technology for encoding genes and plant genomes, applied in the field of rice artificial genome editing system, which can solve problems such as the application limitation of single base editing technology and the absence of NGGPAM sequences.

Pending Publication Date: 2020-12-15
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the demand for NGG PAM sequences also makes the system subject to many limitations in the actual application process, such as the lack of suitable NGG PAM sequences near the target site to be edited, etc.
Especially when using single base editing technology to edit specific target bases, it is necessary to consider both the base editing activity window and the recognition of PAM sequences, which makes the application of single base editing technology more limited.

Method used

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  • Rice artificial genome editing system with expanded editing scope and applications thereof
  • Rice artificial genome editing system with expanded editing scope and applications thereof
  • Rice artificial genome editing system with expanded editing scope and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1, Screening and Identification of Cas9-AA Recognition of PAM Sequence in Rice

[0046] 1. Design of a total of 20 target sequences for 16 genes including OsMPK3 and construction of gene editing vectors

[0047] Select the gene accession numbers and 20 target sequences of the designed OsMPK3 and other 16 genes (according to 5′-N 3 -3' form, carry out the combination of four bases for the last two sites in the PAM, designed 16 kinds of PAM) information is shown in Table 1, the genome sequences of these genes are all from the rice genome database ( https: / / rapdb.dna.affrc.go.jp / ) obtained. Each target sequence (5'-N 19-20 The forward and reverse oligonucleotide chains of PAM-3′) (see Table 1 for the specific sequence) were artificially synthesized by Sangon Bioengineering (Shanghai) Co., Ltd., using T 4 Polynucleotide kinase phosphorylates the primer and anneals to form a double-stranded DNA fragment (containing the 5′-N in the target sequence of the sgRNA 1...

Embodiment 2

[0072] Example 2, Construction of rice cytosine base editor expression vector

[0073] In the rice cytosine base editor expression vector pUbi-rBE66 provided in this example, the expressed cytosine base editor is a fusion protein named rBE66 (i.e. Cas protein / cytidine deaminase), which is named hAID* A protein connected by cytosine deaminase of Δ, Cas protein named Cas9-AA (D10A), uracil DNA glycosylase inhibitor named UGI and nuclear localization signal named NLS. The amino acid sequence of rBE66 is SEQ ID No.6 in the sequence table, the 1-195th position is the amino acid sequence of hAID*Δ, the 196-211th position is the amino acid sequence of the connecting peptide, and the 212-1578th position is Cas9-AA( In the amino acid sequence of D10A), positions 1579-1669 are the amino acid sequence of UGI, and positions 1670-1676 are the amino acid sequence of NLS.

[0074] Cas9-AA in Example 1 is a double nickase, and the 10th amino acid residue of Cas9-AA (double nickase) is replac...

Embodiment 3

[0076] Example 3. Using pUbi:rBE66 to perform C>T substitution on the target base of rice endogenous gene

[0077] The genome sequences of OsCOI2 (Os03g0265500) and OsSERK2 (Os04g0457800) genes were obtained from the rice genome database ( https: / / rapdb.dna.affrc.go.jp / ) obtained.

[0078] For the OsCOI2 gene, select "5'-GTCGACCCTGCACCAGTGGC GGC -3'" (OsCOI2 gene target sequence 1, the underline is the NGC PAM sequence, and the rest of the sequence is 5'-N 19-20 -3') and "5'-GAGCGCGTCGACCCTGCAC CAG -3'" (OsCOI2 gene target sequence 2, the underline is the NAG PAM sequence, and the rest of the sequence is 5'-N 19-20 -3') as the target nucleotide sequence of sgRNA. In order to prepare sgRNA against OsCOI2 gene target sequence 1, the required synthetic oligonucleotide chains are gOsCOI2-F7 (5′-gtgtGTCGACCCTGCACCAGTGGC-3′) / gOsCOI2-R7 (5′-aaacGCCACTGGTGCAGGGTCGAC-3′), in order to prepare sgRNA of OsCOI2 gene target sequence 2, the required synthetic oligonucleotide chains ...

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PUM

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Abstract

The invention discloses a rice artificial genome editing system with expanded editing scope and applications thereof. The applications of a DNA molecule in single base editing in rice are provided. The DNA molecule consists of an expression cassette containing Cas protein and deaminase and an sgRNA expression cassette; the expression cassette containing Cas protein and deaminase expresses fusion protein; the fusion protein contains the Cas protein and the deaminase; the deaminase is cytosine deaminase and / or adenine deaminase; the Cas protein is the protein of which amino acid sequence is at the position 212-1578 of SEQ ID No.6; the sgRNA expression cassette expresses sgRNA; and the target sequence of the sgRNA is 5'-N19-20PAM-3'. The system can be widely applied to the knockour of targetgenes in rice genomes or the directed mutation of single base.

Description

technical field [0001] The invention relates to a set of rice artificial genome editing system with extended editing range and its application. Background technique [0002] Rice (Oryza sativa L.) is the most important food crop in my country and even in the world, and nearly half of the world's population uses rice as a staple food. China is also the country with the highest total rice output in the world, accounting for about 30% of the global total. However, with the continuous growth of the global population and the production process, various diseases of rice seriously restrict the growth and development of rice, affecting the yield and quality of rice, threatening global food security. Therefore, improving the quality of rice, increasing the disease resistance and stress resistance of rice plants, and increasing the yield of rice to ensure a stable supply of food are major issues for the sustainable development of human society. [0003] In recent years, the plant ge...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/82C12N15/63A01H5/00A01H6/46
CPCC12N15/8216C12N9/78C12N9/22C12N15/113C12Y305/04002C12N2310/10
Inventor 周焕斌周雪平徐子妍旷永洁任斌闫大琦严芳
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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