title: Optimizing sgRNA structure to improve CRISPR-Cas9 knockout efficiency.
abstract: BACKGROUND
  Single-guide RNA (sgRNA) is one of the two key components of the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome-editing system. The current commonly used sgRNA structure has a shortened duplex compared with the native bacterial CRISPR RNA (crRNA)-transactivating crRNA (tracrRNA) duplex and contains a continuous sequence of thymines, which is the pause signal for RNA polymerase III and thus could potentially reduce transcription efficiency.
  
  
  RESULTS
  Here, we systematically investigate the effect of these two elements on knockout efficiency and showed that modifying the sgRNA structure by extending the duplex length and mutating the fourth thymine of the continuous sequence of thymines to cytosine or guanine significantly, and sometimes dramatically, improves knockout efficiency in cells. In addition, the optimized sgRNA structure also significantly increases the efficiency of more challenging genome-editing procedures, such as gene deletion, which is important for inducing a loss of function in non-coding genes.
  
  
  CONCLUSIONS
  By a systematic investigation of sgRNA structure we find that extending the duplex by approximately 5 bp combined with mutating the continuous sequence of thymines at position 4 to cytosine or guanine significantly increases gene knockout efficiency in CRISPR-Cas9-based genome editing experiments.
authors.affiliation: Department of Biomedical Sciences, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center El Paso, El Paso, TX, 79905, USA. ying.dang@ttuhsc.edu.
authors.forename: Ying
authors.initials: Y
authors.lastname: Dang
authors.affiliation: Department of Biomedical Sciences, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center El Paso, El Paso, TX, 79905, USA. gengxiang.jia@ttuhsc.edu.
authors.forename: Gengxiang
authors.initials: G
authors.lastname: Jia
authors.affiliation: Department of Biomedical Sciences, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center El Paso, El Paso, TX, 79905, USA. jenniechoi@gmail.com.
authors.forename: Jennie
authors.initials: J
authors.lastname: Choi
authors.affiliation: Department of Biomedical Sciences, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center El Paso, El Paso, TX, 79905, USA. hongming.ma@ttuhsc.edu.
authors.forename: Hongming
authors.initials: H
authors.lastname: Ma
authors.affiliation: Department of Biomedical Sciences, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center El Paso, El Paso, TX, 79905, USA. edgar.anaya@ttuhsc.edu.
authors.forename: Edgar
authors.initials: E
authors.lastname: Anaya
authors.affiliation: Department of Biomedical Sciences, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center El Paso, El Paso, TX, 79905, USA. chunting.ye@ttuhsc.edu.
authors.forename: Chunting
authors.initials: C
authors.lastname: Ye
authors.affiliation: Department of Biomedical Sciences, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center El Paso, El Paso, TX, 79905, USA. premlata.shankar@ttuhsc.edu.
authors.forename: Premlata
authors.initials: P
authors.lastname: Shankar
authors.affiliation: Department of Biomedical Sciences, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center El Paso, El Paso, TX, 79905, USA. haoquan.wu@ttuhsc.edu.
authors.forename: Haoquan
authors.initials: H
authors.lastname: Wu
mesh_terms: D064130:CRISPR-Associated Proteins / Q000235:genetics; D064113:CRISPR-Cas Systems; D002460:Cell Line; D003850:Deoxyribonuclease I / Q000235:genetics; D017353:Gene Deletion; D055786:Gene Knockout Techniques; D006801:Humans; D019169:Jurkat Cells; D009154:Mutation; D012313:RNA / Q000737:chemistry*
chemical_list: D064130:CRISPR-Associated Proteins; D012313:RNA; D003850:Deoxyribonuclease I