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. 2022 Feb 5;21:19. doi: 10.1186/s12934-022-01746-z

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© The Author(s) 2022

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 2 — Optimizing the CRASH protocol targeting adhE gene with gRNA3. a Recombination efficiencies with four different homology arm (HA) designs. The U50D50 HA design carries 50 bp upstream homologous sequence and 50 bp downstream homologous sequence. The U500D50 HA design carries 500 bp upstream homologous sequence and 50 bp downstream homologous sequence. The U50D500 HA design carries 50 bp upstream homologous sequence and 500 bp downstream homologous sequence. The U500D500 HA design carries 500 bp upstream homologous sequence and 500 bp downstream homologous sequence. All are obtained via PCR using the pTarget-adhE plasmid carrying the homology arm as template. The template is completely removed before transforming the PCR products into BL21 cells for gene deletion. The number of colonies formed on the plates were counted by Qpix (Molecular Device) to determine the transformation efficiency. b Gel image of colony PCR results to check successfully deleted colonies. C is the PCR products obtained from non-edited cell. The size of non-edited (WT) and edited (Deleted) is indicated on the side of the gel. c Knockout efficiency and transformation efficiency with varied upstream HA length. The downstream HA length is kept at 500 bp. d Knockout efficiency and transformation efficiency for various lengths of DNA deleted from the BL21 genome. All the efficiencies were obtained with replicate experiments