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| − | <partinfo>BBa_K1150000 AddReview 1</partinfo>
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| − | <I>Peking 2020</I>
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| − | We designed construction of two CRISPR system guided base editor, [https://parts.igem.org/Part:BBa_K3645008 BBa_K3645008] ABE (A → G on binding site), [https://parts.igem.org/Part:BBa_K3645011 BBa_K3645011] CBE (C → T on binding site), see below for the fusion protein overview.
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| − | <html>
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| − | <p>David Liu’s lab created the first base editor in 2016 (Komor et al., 2016) and since then has been trying to
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| − | expand their precision editing capabilities. Base editors make specific DNA base changes and consist of a
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| − | catalytically impaired Cas protein (dCas or Cas nickase) fused to a DNA-modifying enzyme, in this case a
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| − | deaminase. Base changes from C•G-to-T•A are mediated by cytosine base editors (CBEs) and base changes from
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| − | A•T-to-G•C are mediated by adenine base editors (ABEs). How does this work? Through molecular biology teamwork.
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| − | The guide RNA (gRNA) specifies the editing target site on the DNA, the Cas domain directs the modifying enzyme
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| − | to the target site, and the deaminase induces the DNA base change without a DNA double-strand break. But base
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| − | editors aren’t perfect. They may be slow, can only target certain sites, or make only a subset of base
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| − | substitutions. (addgene blog by Susanna Bachle) </p>
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| − | <p>We used the existing plasmids for enzyme digestion and ligation, and ePCR was added to the BioBrick connector.
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| − | After multiple rounds of splicing and assembly, we obtained the ABE and CBE we needed. The schematic diagrams
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| − | are as follows:</p>
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| − | <h1>CBE</h1>
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| − | <p>Until 2016, precise single base changes were only possible through exploiting the homology-directed repair (HDR)
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| − | pathway which occurs in cells at low frequencies and relies on the efficient delivery of donor DNA to facilitate
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| − | repair. Since the development of CRISPR-mediated base editing (BE), these types of repairs can now be done more
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| − | efficiently than before. A base editor precisely changes a single base with an efficiency typically ranging from
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| − | 2575%, while the success of precise change via HDR limited to 0-5%. This blog post covers a brief review of
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| − | different basic BE technologies and their adaptation for plant genome editing. (addgene blog by Guest Blogger) </p>
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| − | <p>In 2016, two independent groups (komor et al., 2016 and Nishida et al., 2016) invented CRISPR base editor by linking
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| − | cytosine deaminase with cas9 cleavage enzyme (ncas9), thus achieving accurate and efficient base rewriting in the
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| − | genome. Ncas9 creates a gap in DNA by cutting only one single strand, thus greatly reducing the possibility of
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| − | harmful insertion deletion. After binding with DNA, CBE deamination of target cytosine (C) into uracil (U) base.
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| − | Later, the resulting U•G pairs were repaired through the cell mismatch repair mechanism to convert the original C•G
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| − | pair into T•A, or reduced to the original C•G through the uracil glycosylase mediated base excision repair. The
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| − | presence of UGI minimizes the second result and increases the production of required T•A base pairs. </p>
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| − | <img src="https://2020.igem.org/wiki/images/b/b4/T--Peking--CBE-5.jpg">
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| − | <h6>(addgene blog by Guest Blogger)</h6>
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| − | <img src="https://2020.igem.org/wiki/images/2/2f/T--Peking--CBE-1.png">
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| − | <img src="https://2020.igem.org/wiki/images/e/e6/T--Peking--CBE-2.png">
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| − | <img src="https://2020.igem.org/wiki/images/6/64/T--Peking--CBE-3.png">
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| − | <img src="https://2020.igem.org/wiki/images/6/6c/T--Peking--CBE-4.png">
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| − | <h1>ABE</h1>
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| − | <img src="https://2020.igem.org/wiki/images/f/f2/T--Peking--ABE-4.jpeg">
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| − | <h6>(Gaudelli et al., 2020.)</h6>
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| − | <p>Gaudelli et al. have successfully developed an adenosine deaminase, which can act on DNA for adenine base editing.
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| − | They first created a defective chloramphenicol resistance gene (CamR) by introducing a point mutation (H193Y).
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| − | Reversal of this mutation by adenine base editor will restore antibiotic resistance. To find such a protein, they
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| − | created a mutant library of E.coli tRNA adenosine deaminase (ecTadA), fused it with dcas9, and transformed it into
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| − | E.coli containing the defective CamR gene. Screening of viable colonies and subsequent rounds of evolution and
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| − | engineering produced a mutant TadA (TadA *), which accepted DNA as a substrate satisfactorily. </p>
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| − | <p>
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| − | The artificially evolved adenosine deaminase catalyzes the transformation of target "A" into "I" (inosine), which is
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| − | regarded as "G" by cell polymerase. Subsequently, a primitive genome A•T base pair was transformed into a G•C base
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| − | pair. Since inosine excision repair is not as active as uracil excision, ABE does not require any additional
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| − | inhibitor proteins, such as UGI in CBE.
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| − | </p>
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| − | <img src="https://2020.igem.org/wiki/images/d/d8/T--Peking--ABE-1.png">
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| − | <img src="https://2020.igem.org/wiki/images/f/f7/T--Peking--ABE-2.png">
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| − | <img src="https://2020.igem.org/wiki/images/f/f7/T--Peking--ABE-3.png">
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| − | <h1>Citation</h1>
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| − | <p>[1] Madej T, Lanczycki CJ, Zhang D, Thiessen PA, Geer RC, Marchler-Bauer A, Bryant SH. " MMDB and VAST+: tracking
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| − | structural similarities between macromolecular complexes. Nucleic Acids Res. 2014 Jan; 42(Database issue):D297-303
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| − | </p>
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| − | <p>[2] Gaudelli NM, Komor AC, Rees HA, Packer MS, Badran AH, Bryson DI, Liu DR. Programmable base editing of A•T to G•C
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| − | in genomic DNA without DNA cleavage. Nature. 2017 Nov 23;551(7681):464-471. doi: 10.1038/nature24644. Epub 2017 Oct
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| − | 25. Erratum in: Nature. 2018 May 2;: PMID: 29160308; PMCID: PMC5726555.</p>
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