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Engineered Cas12i nuclease as well as effect protein and application thereof

A nuclease, engineering technology, used in genetic engineering, hydrolase, biochemical equipment and methods, etc.

Active Publication Date: 2021-07-23
INST OF ZOOLOGY CHINESE ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, current CRISPR-Cas systems have several limitations, including limited gene editing efficiency

Method used

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  • Engineered Cas12i nuclease as well as effect protein and application thereof
  • Engineered Cas12i nuclease as well as effect protein and application thereof
  • Engineered Cas12i nuclease as well as effect protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0320] Example 1: Replace the amino acid that interacts with PAM in the reference Cas12i2 enzyme with a positively charged amino acid, and verify its gene editing efficiency.

[0321] Plasmid construction

[0322] The coding sequence of Cas12i2 was codon optimized (human) and synthesized. Variants of the Cas protein were generated by PCR-based site-directed mutagenesis. The specific method is to divide the DNA sequence design of the Cas12i2 protein into two parts centered on the mutation site, design two pairs of primers to amplify the two parts of the DNA sequence, and introduce the sequence to be mutated into the primers, and finally clone by Gibson Both fragments were loaded into the pCAG-2A-eGFP vector. The combination of mutants is constructed by splitting the DNA of the Cas12i2 protein into multiple segments and using PCR and Gibson clone. The position of the mutant was determined by analyzing the structural information of Cas12i2 using protein structure visualization...

Embodiment 1-A

[0327] Example 1-A Four engineered Cas12i2 with single amino acid substitutions were selected

[0328]According to the method described in Example 1, engineered Cas12i2 enzymes with a single mutation in the amino acid sequence were respectively expressed, and the preferred amino acid replacement mode and its corresponding gene editing efficiency are shown in Figure 1 and Table 1. In Figure 1, we first selected 10 amino acids within 9 Å of Cas12i2 from PAM DNA: E176, E178, Y226, A227, N229, E237, K238, K264, T447, E563, and carried out point mutations of arginine (R) test. By comparing the gene editing efficiency of these mutants and wild-type Cas12i2 at two genomic loci: CCR5-3, RNF2-7 in 293T cells, we found that mutants with the following amino acid substitutions: E176R, K238R, T447R and E563R can effectively improve the gene editing efficiency (shown in Figure 1, there are four amino acid substitutions obtained higher than about 10% (this is the gene editing efficiency of ...

Embodiment 1-B

[0329] Example 1-B compares the engineered Cas12i2 with multiple preferred amino acid substitutions

[0330] According to the method described in Example 1, respectively express the engineered Cas12i2 enzyme whose amino acid sequence has more than two preferred amino acid substitutions, the combination mode and gene editing efficiency comparison are shown in Table 1 and figure 2 . figure 2 It is shown that we combined the point mutations in the four efficiency-improving mutants, E176R, K238R, T447R and E563R, which were screened in Example 1-A. By comparing the gene editing efficiency of these mutants and wild-type Cas12i2 at three genomic loci: CCR5-3, CCR5-5, and RNF2-7 in 293T cells, we found that the efficiency can be further improved after the combination of point mutations mutant. Especially when the four mutations are combined (E176R+K238R+T447R+E563R), the most efficient combined mutation can be obtained.

[0331] The experimental result summary of embodiment 1

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Abstract

The invention provides an engineered Cas12i nuclease, which comprises one, two, three or four of following mutations based on reference Cas12i nuclease: (1) replacing one or more amino acids which interact with PAM in the reference Cas12i nuclease with positively charged amino acids; and / or (2) replacing one or more amino acids which participat in opening DNA double chains in the reference Cas12i nuclease with amino acids with aromatic rings; and / or (3) replacing one or more amino acids which are located in the RuvC structural domain and interact with the single-stranded DNA substrate in the reference Cas12i nuclease with positively charged amino acids; and / or (4) replacing one or more amino acids which interact with the DNA-RNA double helix in the reference Cas12i nuclease with positively charged amino acids. Particularly, the reference Cas12i nuclease is natural Cas12i nuclease, such as natural Cas12i2 nuclease, and the amino acid sequence of the reference Cas12i nuclease is as defined in SEQ ID NO. 1.

Description

technical field [0001] This application belongs to the field of biotechnology. More specifically, the application relates to Cas12i nucleases, effector proteins and uses thereof with improved catalytic activity (eg, gene editing activity). Background technique [0002] Genome editing is an important and useful technique in genome research. Several systems are available for genome editing, including clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems, transcription activator-like effector nuclease (TALEN) systems, and zinc finger nuclease (ZFN) systems. [0003] The CRISPR-Cas system is an efficient and cost-effective genome editing technology that can be widely applied in a range of eukaryotes from yeast and plants to zebrafish and humans (see review: Van der Oost 2013, Science339: 768- 770, and Charpentier and Doudna, 2013, Nature 495: 50-51). The CRISPR-Cas system provides adaptive immunity in archaea and bacteria by combining Cas12i effector p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/22C12N15/113C12Q1/6869
CPCC12N9/22C12N15/113C12Q1/6869C12N2310/20C12Q2535/122C12Q2521/327
Inventor 李伟周琪陈阳灿胡艳萍王鑫阁陈逸
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
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