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To develop a more rigorous and Can1 -independent assay for base editor specificity, we also investigated the worst-case scenario, in which all nucleotides within the BE3 activity window are Cs i. Analysis of editing products by deep sequencing revealed that base editors with 5—7 aa rigid linkers mainly edited at positions C to C Importantly, when editing product distribution was analyzed, BE3-treated sequences mostly contained four simultaneously edited bases, whereas short rigid linker-containing base editors predominantly generate products with one to three edited bases Supplementary Figure 5c , thus providing further evidence for short rigid linkers leading to more precise editing.
To test whether other base editors can also be improved by engineering the linker region connecting the nucleoside deaminase domain with the nCas9 domain, we next applied a similar strategy to CDA1, the AID homolog of sea lamprey 17 that has been reported to exhibit superior performance to APOBEC1 in certain sequence contexts By contrast, when CDA1 was fused to either the N-terminus or the C-terminus of nCas9, both fusions exhibited high editing efficiency.
However, there was a remarkable difference in the width of the editing window, in that the N-terminal CDA1 nCDA1-BE3 triggered editing in a much broader window when tested on either an oligo C substrate or target sites in the Can1 gene Fig. Comparison of N- and C-terminal deaminase fusions to nCas9. The sequence of the target C 9 motif is shown with the numbers representing the position of possible editing targets red relative to the PAM blue.
Supplementary Figure 1 ; Supplementary Table 4. Source data underlying panels b and c are provided as a Source Data file. Comparative assessment of the specificity of previously generated base editors and our base editors on several genomic target sequences showed that, in many cases, some level of discrimination between adjacent Cs is possible, but the achievable precision depends on the sequence context and on the base editor used Supplementary Figure 7.
Moreover, CDA1-based editors enhance product purity Supplementary Figure 8 , as reported previously Surprisingly, both linkerless fusions showed an unaltered activity window with largely unchanged editing efficiency at each C within it Supplementary Figure 9. This result suggests that the termini of CDA1 are inherently flexible and may act as linker-like sequences. We, therefore, tested the impact of N- and C-terminal truncations removing potential linker-like fragments on base editing.
The enzyme tolerated truncations up to amino acid residue without a significant loss in editing efficiency Fig. Larger deletions had similar beneficial effects on editing precision, although some of them displayed slightly reduced overall editing efficiency Fig.
Unlike the full-length base editor, the best-performing truncated variants showed a clear preference for one or two Cs within the oligo C stretch e. Design of base editors with truncated CDA1 domains. Identical amino acid residues are shaded in red, similar residues in yellow. All base editor variants were tested on both C 8 a and C 9 b motifs see Methods. Cs within each target region are shown in red, with the number below indicating their distance from the PAM blue.
Values and error bars represent the mean and standard deviation of three biological replicates. For a comparison with additional deletion constructs, see Supplementary Figures 10 and Tests on oligo C motifs represent the most stringent assays for site selectivity of base editors.
However, such long C stretches would only rarely be targets of genome editing with base editors in vivo. Cytidines representing possible editing targets are shown in red with the subscript number representing their position relative to the PAM blue. We also investigated the indel frequency and base editing purity at these sites when treated by narrowed-window base editors.
We found that the frequency of editing errors was very low, consistent with what has been reported for other base editors Supplementary Figure Finally, we also determined the base editing outcome in individual colonies obtained by the canavanine selection method. Analysis of base editing patterns and efficiencies in single yeast colonies selected for canavanine resistance. Yeast cells were transformed with plasmids expressing the base editor and an sgRNA targeting the Can1—5 site.
The target sequence is shown with the cytidines that can potentially undergo editing in red and the PAM in blue. Values and error bars reflect the mean and standard deviation of three biological replicates. See also Table 1. This did not appreciably affect editing precision, suggesting that the superior performance of our base editors is largely independent of the duration of the exposure of the genome to the base editor Supplementary Table 2.
In this work, we have developed two alternative strategies to effectively reduce the width of the editing window and, in this way, greatly increase the precision of C-to-T base editors. The use of stiff, proline-rich linkers of specific lengths can significantly narrow the editing window and thus improve the accuracy of editing Fig.
We attribute this to a more narrowly defined distance between the nucleoside deaminase domain and the nCas9 domain of the fusion protein that is likely to result in more precise positioning of the deaminase domain on the target sequence. However, our finding that large parts of the C-terminus of CDA1 are dispensable for deaminase function offered the possibility to substantially shorten the distance between the deaminase domain and the nCas9 domain.
Interestingly, this approach resulted in a substantial gain in editing precision and product purity Figs. It is important to note that, in our study, we used the most stringent assays and the worst-case scenarios in that our target sequences of base editing contained multiple cytidines in close proximity or were even entirely comprised of cytidines C 8 or C 9 motifs.
Although our base editors with C-terminally truncated CDA1 domains readily outperformed current base editors, they unsurprisingly, still showed some level of imprecision on these extreme substrates. However, when tested on more normal sequence contexts, our best-performing editors displayed absolute precision in that they i produced only correctly edited clones despite the presence of another C directly adjacent to the target C and ii edited the target sequence with very high efficiency and accuracy in both alleles of the target gene Fig.
Importantly, the strategies described here do not require reduction of the deaminase activity 23 , Thus, our narrow-window base editors combine superior editing precision with high editing efficiency and product purity. To increase the genome-targeting scope, engineered Cas9 variants with altered PAM recognition properties e. Highly precise base editors will be essential for future applications of genome editing in gene therapy, site-directed mutagenesis in vivo, and precision breeding.
A narrower editing window means fewer target nucleotides. Especially for the correction of disease-causing mutations in gene therapy, the introduction of new mutations in the vicinity of the targeted nucleotide position is not tolerable 16 , We, therefore, expect that our high-precision base editors will find wide applications in many areas of basic and applied research.
All primers used in this study are listed in Supplementary Table 3. Cloning and amplification of plasmids were carried out in the E. Y-SUP4t were provided by the laboratory of Dr. The D10A point mutation was introduced into cas9 with primers harboring the desired mutation by amplification of the entire plasmid template followed by Dpn I digestion to remove the parental template. Yeast genomic DNA was extracted according to a published protocol After incubation for 3 days, the colony number on each plate was counted.
Each experiment was performed at least three times on different days. To determine the mutation spectrum, colonies were randomly picked and suspended in sterile water, followed by PCR amplification of the relevant CAN1 fragment and DNA sequencing. Control cultures not treated with base editors did not produce canavanine-resistant colonies. Genomic DNA was extracted from culture samples of 0. The purified index-labeled PCR products were pooled at equal molar ratios.
PCR-free library construction and NGS sequencing, demultiplexing by assigning reads to samples, and data filtering including removal of adaptor sequences, contaminations and low-quality reads from raw reads were done commercially BGI, Hong Kong.
This procedure excluded indel-containing and imperfectly matching reads, and allows summarizing each base calling in an alignment-like manner. In addition, the script calculates the frequencies of all edited products by scanning each aligned read for conversion of the potential target cytidines.
For the analysis of indel frequencies, the sequencing reads were scanned for two exactly matching bp sequences that flank both sides of the region of interest i. Reads without exact matches were excluded from further analysis.
By calculating the length of the region, all sequencing reads exactly matching the length of the reference sequence were classified as not containing an indel, otherwise the read was classified as harboring an indel. Further information on experimental design is available in the Nature Research Reporting Summary linked to this article. The data supporting the findings of this study are available within the paper and its supplementary information files.
A reporting summary for this article is available as a Supplementary Information file. The source data underlying Figs. The original version of this Article contained an error in Figure 5. In the panel c, the right bar chart was inadvertently replaced with a duplicate of the left bar chart. Jinek, M. Science , — Article Google Scholar. Mali, P. RNA-guided human genome engineering via Cas9. Jiang, W. Nekrasov, V. Doudna, J. Science , Wright, A. Cell , 29—44 Komor, A.
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Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Gaudelli, N. Programmable base editing of A. Rees, H. Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery.
Kim, J. Precision genome engineering through adenine and cytosine base editing. Plants 4 , — Zong, Y. Precise base editing in rice, wheat and maize with a Cas9-cytidine deaminase fusion. Liang, P. Protein Cell 8 , — Kim, Y. Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions. Gehrke, J. Zafra, M. Optimized base editors enable efficient editing in cells, organoids and mice.
Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity.