Abstract
Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) is a precise genome manipulating technology that can be programmed to induce double-strand break (DSB) in the genome wherever needed. After nuclease cleavage, DSBs can be repaired by non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathway. For producing targeted gene knock-in or other specific mutations, DSBs should be repaired by the HDR pathway. While NHEJ can cause various length insertions/deletion mutations (indels), which can lead the targeted gene to lose its function by shifting the open reading frame (ORF). Furthermore, HDR has low efficiency compared with the NHEJ pathway. In order to modify the gene precisely, numerous methods arose by inhibiting NHEJ or enhancing HDR, such as chemical modulation, synchronized expression, and overlapping homology arm. Here we focus on the efficiency and other considerations of these methodologies.
Highlights
Reviewed by: James Carney, Sandia National Laboratories (SNL), United States Alex Michael Ward, Sangamo BioSciences, United States
double-strand break (DSB) can be repaired by non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathway
The first step in target recognition is the transient binding of Cas9 to protospacer adjacent motif (PAM) sequences within the target DNA, which promotes the unwinding of the two DNA strands immediately upstream of the PAM (Sternberg et al, 2014), the spacer sequence of the CRISPR RNAs (crRNAs) binds with the unwinded DNA (6–8 bp in length), forms an RNA-DNA heteroduplex and triggers cleavage at the targeted site (Sternberg et al, 2014; Szczelkun et al, 2014)
Summary
Mingjie Liu 1, Saad Rehman 1, Xidian Tang 1, Kui Gu 1, Qinlei Fan 2, Dekun Chen 1* and Wentao Ma 1*. Clustered regularly interspaced short palindromic repeats (CRISPR) represents a family of DNA sequences in bacteria and archaea (Barrangou, 2015) This family is characterized by direct palindromic repeats, where sequences are the same in both directions, varying in size from 21 to 37 bp (Barrangou and Marraffini, 2014), interspaced by spacers, which have fragments gathered from viruses or phages that previously tried to infect the cell (Horvath and Barrangou, 2010; Morange, 2015). The type II CRISPR-Cas system needs only cas to execute immunity in the presence of an existing targeting spacer sequence (Sapranauskas et al, 2011) It requires two small RNAs: the crRNA and the transencoded crRNA(tracrRNA) (Deltcheva et al, 2011). The CRISPR-Cas system introduces a crRNAspecific DSB in the target sequence, which is further resolved either by homology-directed repair (HDR) or non-homologous end joining (NHEJ)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
