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David Liu’s lab created the first base editor in 2016 (Komor et al., 2016) and since then has been trying to expand their precision editing capabilities. Base editors make specific DNA base changes and consist of a catalytically impaired Cas protein (dCas or Cas nickase) fused to a DNA-modifying enzyme, in this case a deaminase. Base changes from C•G-to-T•A are mediated by cytosine base editors (CBEs) and base changes from A•T-to-G•C are mediated by adenine base editors (ABEs). How does this work? Through molecular biology teamwork. The guide RNA (gRNA) specifies the editing target site on the DNA, the Cas domain directs the modifying enzyme to the target site, and the deaminase induces the DNA base change without a DNA double-strand break. But base editors aren’t perfect. They may be slow, can only target certain sites, or make only a subset of base substitutions. (addgene blog by Susanna Bachle)
We used the existing plasmids for enzyme digestion and ligation, and ePCR was added to the BioBrick connector. After multiple rounds of splicing and assembly, we obtained the ABE and CBE we needed. The schematic diagrams are as follows:
CBE
Until 2016, precise single base changes were only possible through exploiting the homology-directed repair (HDR) pathway which occurs in cells at low frequencies and relies on the efficient delivery of donor DNA to facilitate repair. Since the development of CRISPR-mediated base editing (BE), these types of repairs can now be done more efficiently than before. A base editor precisely changes a single base with an efficiency typically ranging from 2575%, while the success of precise change via HDR limited to 0-5%. This blog post covers a brief review of different basic BE technologies and their adaptation for plant genome editing. (addgene blog by Guest Blogger)
In 2016, two independent groups (komor et al., 2016 and Nishida et al., 2016) invented CRISPR base editor by linking cytosine deaminase with cas9 cleavage enzyme (ncas9), thus achieving accurate and efficient base rewriting in the genome. Ncas9 creates a gap in DNA by cutting only one single strand, thus greatly reducing the possibility of harmful insertion deletion. After binding with DNA, CBE deamination of target cytosine (C) into uracil (U) base. Later, the resulting U•G pairs were repaired through the cell mismatch repair mechanism to convert the original C•G pair into T•A, or reduced to the original C•G through the uracil glycosylase mediated base excision repair. The presence of UGI minimizes the second result and increases the production of required T•A base pairs.
ABE
Gaudelli et al. have successfully developed an adenosine deaminase, which can act on DNA for adenine base editing. They first created a defective chloramphenicol resistance gene (CamR) by introducing a point mutation (H193Y). Reversal of this mutation by adenine base editor will restore antibiotic resistance. To find such a protein, they created a mutant library of E.coli tRNA adenosine deaminase (ecTadA), fused it with dcas9, and transformed it into E.coli containing the defective CamR gene. Screening of viable colonies and subsequent rounds of evolution and engineering produced a mutant TadA (TadA *), which accepted DNA as a substrate satisfactorily.
The artificially evolved adenosine deaminase catalyzes the transformation of target "A" into "I" (inosine), which is regarded as "G" by cell polymerase. Subsequently, a primitive genome A•T base pair was transformed into a G•C base pair. Since inosine excision repair is not as active as uracil excision, ABE does not require any additional inhibitor proteins, such as UGI in CBE.
gRNA-Scaffold
EvolvR
1. Introduction of EvolvR
The capacity to diversify genetic codes advances our ability to understand and engineer biological systems. Several methods for generating mutant libraries exist, but none provide a means to continuously diversify all nucleotides within a user-defined genomic region. EvolvR, a CRISPR-Cas9 based targeted mutagenesis method developed by the Dueber Lab at Berkeley, provides a new approach for generating novel genetic variants in bacteria.
By fusing error prone polymerase Poll3M to nCas9, the nick site, introduced by sgRNA targeting and nCas9 nicking, serves as the initiate point for error prone PolI3M function. Due to its modular nature, the Dueber Lab created a few versions of EvolvR. PolI5M, generated by two additional mutations to PolI3M which increased EvolvR's mutagenesis rate to ~10-3 mutations per nucleotide per generation.
Here we use PolI5M and eSpnCas9 to target the music sequence, expecting to generate mutated music sequence and "listen to the mutation".
2. Construction of EvolvR
We use GoldenGate Assembly to construct EvolvR. By synthesizing eSpnCas9-linker-PolI5M 1kb per 1kb, we add GoldenGate tag and linked the parts. After that, the part was linked to standard pSB1C3 vector by T4 DNA ligase mediated ligation.
3. Experimental progress of EvolvR
Constructing pEvolvR
Ligating EvolvR
Cutting pSB1C3 vector
Linking EvolvR and pSB1C3 vector
Introduce pEvolvR to bacteria
Sequencing for the editing outcome
Citation
[1] Madej T, Lanczycki CJ, Zhang D, Thiessen PA, Geer RC, Marchler-Bauer A, Bryant SH. " MMDB and VAST+: tracking structural similarities between macromolecular complexes. Nucleic Acids Res. 2014 Jan; 42(Database issue):D297-303
[2] Gaudelli NM, Komor AC, Rees HA, Packer MS, Badran AH, Bryson DI, Liu DR. Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature. 2017 Nov 23;551(7681):464-471. doi: 10.1038/nature24644. Epub 2017 Oct 25. Erratum in: Nature. 2018 May 2;: PMID: 29160308; PMCID: PMC5726555.
[3] Banno S, Nishida K, Arazoe T, Mitsunobu H, Kondo A. Deaminase-mediated multiplex genome editing in Escherichia coli. Nat Microbiol. 2018 Apr;3(4):423-429. doi: 10.1038/s41564-017-0102-6. Epub 2018 Feb 5. PMID: 29403014.