Abstract
The CRISPR/Cas12a (Cpf1) system utilizes a thymidine-rich protospacer adjacent motif (PAM) and generates DNA ends with a 5′ overhang. These properties differ from those of CRISPR/Cas9, making Cas12a an attractive alternative in the CRISPR toolbox. However, genome editing efficiencies of Cas12a orthologs are generally lower than those of SpCas9 and depend on their target sequences. Here, we report that the efficiency of FnCas12a-mediated targeted mutagenesis varies depending on the length of the crRNA guide sequence. Generally, the crRNA of FnCas12a contains a 24-nt guide sequence; however, some target sites showed higher mutation frequency when using crRNA with an 18-nt or 30-nt guide sequence. We also show that a short crRNA containing an 18-nt guide sequence could induce large deletions compared with middle- (24-nt guide sequence) and long- (30-nt guide sequence) crRNAs. We demonstrate that alteration of crRNA guide sequence length does not change the rate of off-target mutation of FnCas12a. Our results indicate that efficiency and deletion size of FnCas12a-mediated targeted mutagenesis in rice can be fine-tuned using crRNAs with appropriate guide sequences.
Highlights
The CRISPR/Cas9 system was first reported as an adaptive immune system in archaea and bacteria and is used for genome editing in various organisms, including plants (Li et al, 2013; Nekrasov et al, 2013; Shan et al, 2013)
FnCas12a/CRISPR RNA (crRNA) vectors were transformed into rice calli via A. tumefaciens strain EHA105, and mutations were detected by cleaved amplified polymorphic sequences (CAPS) analysis (Figure 1)
When DROOPING LEAF (DL)-1_Short was used, undigested DNA fragments were clearly detected in all transgenic calli, and mutation frequencies at the DL-1 target site were higher than that of DL-1_Middle (Figure 1A, upper panel)
Summary
The CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) system was first reported as an adaptive immune system in archaea and bacteria and is used for genome editing in various organisms, including plants (Li et al, 2013; Nekrasov et al, 2013; Shan et al, 2013). In CRISPR/Cas9-mediated genome editing, the PAM restricts the selectivity of target sites because each Cas requires a specific crRNA Length Affects CRISPR/FnCas12a Mutagenesis. Engineered SpCas variants that recognize different PAM sequences have been developed, expanding the application of genome editing in plants (Hu et al, 2018; Meng et al, 2018; Endo et al, 2019). These Cas orthologs and variants can expand target selectivity. Our results suggest that optimizing target length can lead to more efficient CRISPR/FnCas12a-mediated genome editing in plants
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