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Gene editing method based on gene cas3 of I-B type CRISPR-Cas system

A gene editing and gene technology, applied in the field of gene editing in the field of biotechnology, can solve the problems of off-target, large molecular weight, etc., and achieve the effect of small molecular weight and rapid gene editing

Pending Publication Date: 2018-01-09
ANHUI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Since 2 types of Cas9 (4107nt) (http: / / www.ncbi.nlm.nih.gov), CjCas9 (2955nt) (E.Kim, T.Koo, S.W.Park, D.Kim, K.Kim, H.Y.Cho, D.W.Song, K.J.Lee, M.H.Jung, S.Kim, J.H.Kim, J.S.Kim, In vivo genome editing with a small Cas9orthologue derived from Campylobacter jejuni. Nat Commun 2017, (8) 14500) with type 2 Cpf1 (3924nt) (R.D.Fagerlund, R.H.Staals, P.C. Fineran, The Cpf1 CRISPR-Cas protein expands genome-editing tools. Genome Biol 2015, (16) 251) has a large molecular weight, and many carriers (such as viruses, vectors, etc.) can only carry limited The length of the fragment, and there are deficiencies such as off-target

Method used

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  • Gene editing method based on gene cas3 of I-B type CRISPR-Cas system
  • Gene editing method based on gene cas3 of I-B type CRISPR-Cas system
  • Gene editing method based on gene cas3 of I-B type CRISPR-Cas system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 Knockout of lacZ gene in EC JM109 (DE3)

[0055] (1) Construction of Cas3 expression vector pCas-cas3

[0056]According to the DNA sequence information of the SV IBL14 gene cas-3 and the vector pCas, the specific forward primer pCas-cas3-F and the reverse primer pCas-cas3-R with the gene cas3 complementary to the vector pCas were designed. Extract SV IBL14 genomic DNA, and amplify the gene cas3 by conventional PCR. The PCR reaction system (50 μL) includes: 5 μL 10×Pfu buffer, 5 μL dNTPs (2.5 mM each), 1 μL 10 μM cas3-F, 1 μL 10 μM cas3-R, 5 μL DMSO, 0.5μL Pfu DNAPolymerase, 0.5μL SV IBL14 genomic DNA, 32μL sterile water (nuclease-free), reaction conditions: 95℃ for 5min, 94℃ for 60±30s, 55±3℃ for 30s, 72℃ for 90±30s, 2.5±0.5 U DNA polymerase (TransStartFastPfu DNA Polymerase, Quanshijin Biotechnology Co., Ltd.), 30 cycles, 72°C for 10min. The PCR product was detected by 1% agarose electrophoresis, recovered by the kit, and the purified full-length cas3 gene ...

Embodiment 2

[0079] Example 2 Knockout of ldh gene and insertion of chloramphenicol resistance gene cat in BS 168

[0080] (1) Construction of Cas3 expression vector pHT304-cas3

[0081] According to the DNA sequence information of SV IBL14 gene cas3 and carrier pHT304, design has the specific forward primer and reverse primer PHT304-cas3-F / R of the gene cas3 complementary to carrier pHT304, all the other steps are with embodiment 1 step (1 ).

[0082] (2) Construction of gene editing vector pKC1139-t / g-Δldh::cat

[0083] Design g-Δldh according to the BS 168 gene ldh sequence and design t-Δldh::cat inserted between the upper and lower homology arms of the ldh gene to express the chloramphenicol resistance enzyme gene cat sequence, and the remaining steps are the same as in Example 1 ( 2).

[0084] The synthetic t-Δldh::cat sequence is shown in Table 4, in which the single underline represents the promoter; the double underline metabolic terminator.

[0085] Table 4 t-Δldh::cat sequenc...

Embodiment 3

[0100] Example 3 Double plasmid knockout of SC BY4741crtE gene

[0101] (1) Construction of Cas3 expression vector pRS415-cas3

[0102] In addition to the DNA sequence information of the vector pRS415, design the specific forward primer and reverse primer pRS415-cas3-F / R with the gene cas3 complementary to the vector pRS415, and the upstream of the gene cas3 is the gal1 promoter, and the downstream end has a nucleus The signal sequence and the CYC1 terminator are located, and the rest of the steps are the same as step (1) in Example 1.

[0103] (2) Construction of gene editing vector pYES2-NTA-t / g-ΔcrtE

[0104] Except for designing g-ΔcrtE and designing and synthesizing t-ΔcrtE according to the SC BY4741crtE gene sequence, other steps are the same as step (2) of Example 1.

[0105] The synthesized t-ΔcrtE sequence is shown in Table 7.

[0106] Table 7 t-ΔcrtE sequence list

[0107]

[0108] The synthesized g-ΔcrtE sequence is shown in Table 8. Capital letters in the ta...

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Abstract

The invention discloses a novel gene editing system which is established based on gene cas3 of an I-B type CRISPR-Cas system in a chromosome of Virginia streptomycete IBL14, the gene editing of an I type CRISPR-Cas system to a biological cell genome is realized for the first time, and new supplement and choice are provided for the gene editing system which is established by II type commercializedCas9. In the system, a target DNA can be specifically cut by the Cas3 through crRNA guide or t-DNA location. By adopting the system, error-free, simple and rapid gene editing can be performed on prokaryotic and eukaryotic genomes. The optimized gene editing system is expected to be superior to the commercialized gene editing system which is established based on Cas9 in multiple fields due to low molecular weight and ability to be guided by DNA.

Description

technical field [0001] The present invention relates to the gene editing technology in the field of biotechnology, specifically a gene editing method based on the I-B type CRISPR-Cas system gene cas3. Background technique [0002] CRISPR-Cas systems are ubiquitously present in archaeal and bacterial genomes. The currently discovered CRISPR-Cas systems are divided into two types and six types: type 1 is the interference of multiple Cas proteins, including types I, III, and IV; type 2 is the interference of single Cas proteins, including types II, V, and VI; To type VI, Cas3, Cas9, Cas10, undetermined, Cpf1 and C2c2 were used as marker proteins (P.Mohanraju, K.S.Makarova, B.Zetsche, F.Zhang, E.V.Koonin, J.van der Oost, Diverse evolutionary roots and mechanistic variations of the CRISPR-Cassystems. Science 2016, (353) 5147). Among them, type I is the most widespread, accounting for more than 60% of the identified ones. Unfortunately, although many discoveries have been made ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/90
CPCC12N2310/20C12N2800/80C12N9/22C12N15/113C12N15/907C12N15/11
Inventor 童望宇唐严严夏婷婷
Owner ANHUI UNIVERSITY
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