MMEJ-KO: a web tool for designing paired CRISPR guide RNAs for microhomology-mediated end joining fragment deletion
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EJ-KO: a web tool for designing paired CRISPR guide RNAs for microhomology-mediated end joining fragment deletion 1,2*
Xianrong Xie 1
1,2
1
1,2
, Weizhi Liu , Gang Dong , Qinlong Zhu
& Yao-Guang Liu
1,2*
State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Life Sciences, South China Agricultural University, Guangzhou 510642, China; Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou 510642, China
2
Received June 29, 2020; accepted August 11, 2020; published online September 24, 2020
Citation:
Xie, X., Liu, W., Dong, G., Zhu, Q., and Liu, Y.G. (2020). MMEJ-KO: a web tool for designing paired CRISPR guide RNAs for microhomologymediated end joining fragment deletion. Sci China Life Sci 63, https://doi.org/10.1007/s11427-020-1797-3
Dear Editor, Clustered regularly interspaced short palindromic repeats associated protein (CRISPR/Cas) systems are convenient and versatile tools for genome editing. Directed by a guide RNA (gRNA), the Cas nuclease produces double-strand break (DSB) at the target site, generally resulting in small base insertion/deletion mutations by the non-homologous end joining (NHEJ) DNA repair. However, such small mutations may frequently generate unexpected, abnormal genetic effects that puzzle functional analysis (Tuladhar et al., 2019). CRISPR/Cas tools have also been used to study noncoding genomic elements, such as regulatory regions and non-coding RNAs (Ho et al., 2015). However, it’s often hard to deprive the functions of non-coding elements by small mutations. Instead, the strategy using pairs of gRNAs to generate fragment deletions has been pursed. However, the efficiency of NHEJ-mediated fragment deletions is relatively low (Tan et al., 2020). Microhomology-mediated end joining (MMEJ) is another DNA repair pathway, which depends on recombination between microhomologous sequences (MHSs, 4–25 bp) that flank DSBs (Decottignies, 2007). Recent studies have shown that MMEJ can induce fragment deletions with higher efficiencies (Owens et al., 2019; Tan et al., 2020). *Corresponding authors (Yao-Guang Liu, email: [email protected]; Xianrong Xie, email: [email protected])
In spite that numerous software tools have been developed for designing editing targets (Hanna and Doench, 2020), a dedicated tool for designing MMEJ-based fragment deletion is absent. Thus, development of such a tool will greatly facilitate researchers in various biological studies where deletion of target sequences by genome editing is needed. To generate MMEJ-fragment deletion in a target region, the Cas-cleavage sites are expected to be located near to the inner sides of paired MHSs (Figure 1A). The resultant DSBs may induce MMEJ event by annealing and repairing between the MHSs, thus producing a fragment deletion. Usually, the resulting DSBs close as possible to the MHSs will increase the chance of MHS-recombination, thus producing fragment deletion more efficiently (Tan et al., 2020). Based on this principle, we developed a webbased tool, MMEJ-KO (http://
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