Web-Based Base Editing Toolkits: BE-Designer and BE-Analyzer
The CRISPR-Cas system is broadly used for genome editing because of its convenience and relatively low cost. However, the use of CRISPR nucleases to induce specific nucleotide changes in target DNA requires complex procedures and additional donor DNAs. Fu
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Introduction CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR associated) effectors, naturally used as an adaptive immune system in bacteria and archaea for targeting viral nucleic acid sequences, have been applied for genome editing due to their convenience and high efficacy [1–5]. CRISPR-Cas nucleases recognize protospacer-adjacent motif (PAM) sequences and induce DSBs in target regions in a guide RNA-dependent manner [6]. The DNA DSBs are typically repaired by the cell’s own repair pathways: nonhomologous end joining (NHEJ) or homology-directed repair (HDR) [7]. NHEJ occurs throughout the cell cycle but the repair process is frequently accompanied by errors such as small insertions or deletions (indels) [8]. In contrast, HDR occurs mostly during G2 and S phases, and corrects DNA DSBs precisely without causing mutations but with relatively lower
Mario Andrea Marchisio (ed.), Computational Methods in Synthetic Biology, Methods in Molecular Biology, vol. 2189, https://doi.org/10.1007/978-1-0716-0822-7_7, © Springer Science+Business Media, LLC, part of Springer Nature 2021
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Gue-Ho Hwang and Sangsu Bae
efficiency [9]. For these reasons, NHEJ and HDR have dominantly been used for introducing target gene knockouts and knock-ins, respectively [10–12]. Hence, for inducing a specific nucleotide correction in a target region, the applicability of CRISPR-Cas nucleases is limited; additional donor DNAs are necessary, the editing efficiency is not high enough, and DNA in nondividing cells is not editable. The advent of cytosine and adenine base editors, respectively called CBEs (for C-to-T conversions) and ABEs (for A-to-G conversions), has enabled the correction of many pathogenic genetic variants, with high efficacy in the absence of any donor DNA and without the generation of DNA DSBs. The first to be developed, CBEs were initially created by combining dCas9 or nCas9 with a cytidine deaminase such as rAPOBEC1, PmCDA1, or hAPOBEC3. ABEs were later constructed in a similar manner, by fusing an adenine deaminase to dCas9 or nCas9. In this case, because an adenine deaminase that accepts single-stranded DNA (ssDNA) as a substrate is unknown in nature, ssDNA-targetable enzymes were obtained by evolving an Escherichia coli adenine deaminase, TadA, from its natural function of targeting transfer RNAs (tRNAs) to targeting ssDNAs. Recently, side effects of CBEs and ABEs, such as off-target single-nucleotide conversions, have been reported [13– 17]. Several newer versions of both CBEs and ABEs were developed to improve specificities or efficacies, and to decrease the size of the target window: these include BE4 [18], BE4max [19], EvoFERNY-BE4max [20], ABEmax [21], ABE7.10 [22], and miniABEmax [23]. In addition to DNA base editors, RNA base editors, which are constructed by using CRISPR-Cas13b nucleases that target RNAs instead of DNAs, have been reported [24]. In contrast to the original CRISPR-Cas nucleases, CBEs and ABEs can respectively convert multiple cytidines and adenosines within
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