Optimized LbCas12a variant efficiently generates homozygous or biallelic mutations in Arabidopsis grown at 22 °C in a single generation

This study is led by Professor Qi-Jun Chen (State Key Laboratory of Plant Environmental Resilience, College of Biological Sciences, China Agricultural University, Beijing 100193, China). The authors generated five Cas12a (Cpf1) variants by combining the mutations from the low-temperature-tolerant variants ttAsCas12a (E174R) and ttLbCas12a (D156R), and those from the highly active variants AsCas12a Ultra (M537R and F870L) and LbCas12a Ultra (E795L). The authors conducted parallel tests for five Cas12a variants: LbCas12a, ttLbCas12a (D156R), ttLbCas12a Ultra (D156R, E795L), AsCas12a Ultra (M537R, F870L), and ttAsCas12a Ultra (E174R, M537R, F870L) at six target sites of four genes in Arabidopsis (Arabidopsis thaliana). The authors demonstrated that the variant ttLbCas12a Ultra, harboring the D156R and E795L mutations, exhibited the highest editing efficiency of all variants tested in Arabidopsis and can be used to generate homozygous or biallelic mutants in a single generation in Arabidopsis plants grown at 22°C.

The authors optimized ttLbCas12a Ultra by varying nuclear localization signal sequences and codon usage and found that the optimization further greatly improved editing efficiency. To test whether mutations are heritable, the authors isolated T-DNA-free T2 seeds from T1 homozygous or biallelic mutant plants. The authors demonstrate that mutations present in the T1 plants are heritable. High editing efficiency usually means high off-target mutagenesis. The authors selected the most efficient two targets to analyze off-target mutations. The authors detected no off-target mutations, indicating that careful selection of targets will be able to avoid off-target mutagenesis induced by highly efficient LbCas12a variants.

In conclusion, a LbCas12a variant, ttLbCas12a Ultra, harboring the D156R and E795L mutations from a low-temperature-tolerant variant and a highly active variant, respectively, achieved high editing efficiency in Arabidopsis grown at 22°C; ttLbCas12a Ultra can be further optimized by varying nuclear localization signal sequences and codon usage; ttLbCas12a Ultra may thus be used to efficiently generate homozygous or biallelic mutants in a single generation in Arabidopsis grown at 22°C, providing a valuable alternative to Cas9 for editing genes or promoters in Arabidopsis.

See the article:

Enhanced editing efficiency in Arabidopsis with a LbCas12a variant harboring D156R and E795L mutations

https://link.springer.com/article/10.1007/s42994-024-00144-w