CRISPR-Cas9 Gene Deletion Protocol

All constructs used in the study are listed in Additional file 2: Table S1. Sequences of the primers, crRNAs, and oligonucleotides used in the study are listed in Additional file 3: Table S2. Plasmids and chromosomal DNA were extracted using AxyPrep kits (Corning, China). DNA polymerases used were PrimerSTAR (Takara, Japan) or Taq DNA polymerases (Tsingke, China). Restriction endonucleases and T4 DNA ligase were from Thermo Scientific (USA), and the isothermal assembly method was used in this work [35 (link)]. Gene deletions were confirmed by PCR and Sanger sequencing (Tsingke, China).

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Shen W., Zhang J., Geng B., Qiu M., Hu M., Yang Q., Bao W., Xiao Y., Zheng Y., Peng W., Zhang G., Ma L, & Yang S. (2019). Establishment and application of a CRISPR–Cas12a assisted genome-editing system in Zymomonas mobilis. Microbial Cell Factories, 18, 162.

Publication 2019

PrimerSTAR Restriction endonucleases T4 DNA ligase Sanger sequencing

Chromosomal Dna polymerases Gene deletions Oligonucleotides Plasmids Primers Restriction endonucleases T4 dna ligase Taq dna polymerases

Corresponding Organization :

Other organizations : Hubei University

Top 5 similar protocols

Variable analysis

independent variables

Use of different DNA polymerases (PrimerSTAR or Taq DNA polymerases)
Use of different restriction endonucleases and T4 DNA ligase
Use of the isothermal assembly method

dependent variables

Gene deletions

control variables

Plasmids and chromosomal DNA were extracted using AxyPrep kits
Gene deletions were confirmed by PCR and Sanger sequencing

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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