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

Cas12a (Cpf1), a Class 2 Type V CRISPR/Cas nuclease, has several unique attributes for genome editing and may provide a valuable alternative to Cas9. However, a low editing efficiency due to temperature sensitivity and insufficient cleavage activity of the Cas12a nuclease are major obstacles to its broad application. In this report, we generated two variants, ttAsCas12 Ultra and ttLbCas12a Ultra harboring three (E174R, M537R, and F870L) or two (D156R and E795L) mutations, respectively, by combining the mutations from the temperature-tolerant variants ttAsCas12a (E174R) and ttLbCas12a (D156R), and those from the highly active variants AsCas12a Ultra (M537R and F870L) and LbCas12a Ultra (E795L). We compared editing efficiencies of the five resulting Cas12a variants (LbCas12a, ttLbCas12a, ttLbCas12a Ultra, AsCas12a Ultra, and ttAsCas12 Ultra) at six target sites of four genes in Arabidopsis (Arabidopsis thaliana). 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. In addition, optimization of ttLbCas12a Ultra, by varying nuclear localization signal sequences and codon usage, further greatly improved editing efficiency. Collectively, our results indicate that ttLbCas12a Ultra is a valuable alternative to Cas9 for editing genes or promoters in Arabidopsis. Supplementary Information The online version contains supplementary material available at 10.1007/s42994-024-00144-w.

Table S2.Mutation efficiency of the five Cas12a variants at the GL1-1 and GL1-2 target sites Table S3.Mutation efficiency of three U6 cassettes The mutation efficiency was calculated based on the ratio between the number of plants with mutations to the total number of transgenic plants.When ≥ 95% high-throughput sequencing reads from a line presented the same type of mutation, this line was scored as a homozygous (Ho) mutant; When ≥ 95% high-throughput sequencing reads from a given line presented more than one type of mutation, this line was considered biallelic (Bi).For non-homozygous and non-biallelic mutants, when ≥ 45% or < 45% high-throughput sequencing reads from a line presented the same type of mutation, these lines were scored as heterozygous (He) or chimeric (Chi) mutants, respectively.The data set from U6-tGly is the same as that in Table S1.
Table S4.Mutation efficiency of the three U6 cassettes at the GL1-1 and GL1-2 target sites Mutations in each line was determined by the completely or partially glabrous phenotypes and the mutation efficiency was calculated by the ratio of the number of phenotypically homozygous (Ho), biallelic (Bi), or chimeric (Chi) mutant plants to the total number of transgenic plants.The data set from U6-tGly is the same as that in Table S2.
Table S5.Mutation efficiency of different promoters driving the expression of the crRNA Mutations in each line was determined by the completely or partially glabrous phenotypes and the mutation efficiency was calculated by the ratio of the number of phenotypically homozygous (Ho), biallelic (Bi), or chimeric (Chi) mutant plants to the total number of transgenic plants.The data from U6-tGly is the same as that in Table S2.
Table S6.Mutation efficiency of the two versions of ttLbCas12a Ultra The mutation efficiency was calculated based on the ratio between the number of plants with mutations to the total number of transgenic plants.When ≥ 95% high-throughput sequencing reads from a line presented the same type of mutation, this line was scored as a homozygous (Ho) mutant; When ≥ 95% high-throughput sequencing reads from a given line presented more than one type of mutation, this line was considered biallelic (Bi).For non-homozygous and non-biallelic mutants, when ≥ 45% or < 45% high-throughput sequencing reads from a line presented the same type of mutation, these lines were scored as heterozygous (He) or chimeric (Chi) mutants, respectively.Mutations in GL1-2 in each line was determined by the completely or partially glabrous phenotypes and the mutation efficiency was calculated by the ratio of the number of phenotypically homozygous (Ho), biallelic (Bi), or chimeric (Chi) mutant plants to the total number of transgenic plants.The data of GL1-2 with ttLbCas12a Ultra is the same as that of GL1-2 with U6 in Table S5.
Table S7.Heritable mutations at the ECA3-1, ECA3-2, and GL2 target sites in T-DNA-free T2 plants These T-DNA-free T2 plants were derived from T1 plants harboring ttLbCas12a Ultra and the U6-tRNA cassette.
Table S8.Analysis of mutations at the GL2 target site in T-DNA-free T2 plants  Table S9.Heritable mutations at the TT4 target site in T-DNA-free T2 plants These T-DNA-free T2 plants were derived from T1 plants harboring ttLbCas12a Ultra and the U6-tRNA cassette.
Table S10.Heritable mutations at the GL1-1 and GL1-2 target sites in T-DNA-free T2 plants These T-DNA-free T2 plants were derived from T1 plants harboring ttLbCas12a Ultra and the U6-tRNA cassette.

Table S1 .
Mutation efficiency of the five Cas12a variants at four target sites for three genes

Table S2 .
Mutation efficiency of the five Cas12a variants at the GL1-1 and GL1-2 target sites

Table S4 .
Mutation efficiency of the three U6 cassettes at the GL1-1 and GL1-2 target sites

Table S5 .
Mutation efficiency of different promoters driving the expression of the crRNA designed for the GL1-2 target site

Table S6 .
Mutation efficiency of the two versions of ttLbCas12a Ultra

Table S8 .
Analysis of mutations at the GL2 target site in T-DNA-free T2 plants

Table S9 .
Heritable mutations at the TT4 target site in T-DNA-free T2 plants

Table S10 .
Heritable mutations at the GL1-1 and GL1-2 target sites in T-DNA-free T2 plants

Table S12 .
Analysis of mutations at the GL1-2 target site in T-DNA-free T2 plants

Table S14 . Primer sequences used in this studyTable S14 .
Primer sequences used in this study

Table S15 . Sequences of target sitesTable S15 .
Sequences of target sites

Table S16 . List of Cas12a vectors generated in this studyTable S16 .
List of Cas12a vectors generated in this study