Additional file 1 of Cas12a-mediated gene targeting by sequential transformation strategy in Arabidopsis thaliana
Creators: name: Li, Jing
name: Wei, Qi
name: Cheng, Yiqiu
name: Kong, Dali
name: Kong, Zhe
name: Ke, Yongping
name: Dang, Xiaofei
name: Zhu, Jian-Kang
name: Shimada, Hiroaki
name: Miki, Daisuke
Publisher: name: figshare
Year: 2024
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 1: Supplementary Table S1. Prediction scores for crRNAs. The crRNAs activities were predicted by using CRISPOR online website ( http://crispor.tefor.net/ ). Out-of-Frame score, only for deletions. Predicts the percentage of clones that will carry out-of-frame deletions, based on the micro-homology in the sequence flanking the target site. Supplementary Table S2. Sequences of primers. Supplementary Figure S1. crRNA design in the intergenic region for parental lines. The crRNA was designed at the intergenic region where the two genes, At1g53990 and At1g54000, are located in the tail-to-tail direction. The black arrows indicate the primers used for T7EI digestion assay. The red underline marks crRNA, and bold red represents PAM. Supplementary Figure S2. Evaluation of Cas12a candidate parental lines in T1. A, B, C, D, Bar chart of mutation frequency at the target intergenic region and copy number of Cas12a (A), dCas12a (B), UCas12a (C) and UdCas12a (D) T1 individual candidate parental lines. The histogram is plotted in order of mutation frequency. The white columns represent the mutation frequency at the target intergenic region, the gray columns represent the Cas12a copy number. Asterisks and yellow columns indicate plant lines used as parental lines for sequential transformation. The mutation frequencies of the target intergenic region in the parental lines are indicated. E, F, G, H, Correlation analysis of mutation frequency at the target intergenic region and copy number of Cas12a (E), dCas12a (F), UCas12a (G) and UdCas12a (H). The filled circles represent independent parental lines. The T7EI digestion assay was used to detect the mutation frequencies of intergenic targeting site. The Cas12a copy number was determined by q-PCR in each T1 parental line plants and calculated by the 2−∆∆CT method. Actin7 was used as an internal reference. Supplementary Figure S3. Evaluation of ttCas12a candidate parental lines in T1. A, B, C, D, Bar chart of mutation frequency at the target intergenic region and copy number of ttCas12a (A), dttCas12a (B), UttCas12a (C) and UdttCas12a (D) T1 individual parental line. The histogram is plotted in order of mutation frequency. The white columns represent the mutation frequency at the target intergenic region, the gray columns represent the ttCas12a copy number. Asterisks and yellow columns indicate plant lines used as parental lines for sequential transformation. The mutation frequencies of the target intergenic region in the parental lines are indicated. E, F, G, H, Correlation analysis of mutation frequency at the target intergenic region and ttCas12a copy number in ttCas12a (E), dttCas12a (F), UttCas12a (G) and UdttCas12a (H). The filled circles represent different parental lines. The T7EI digestion assay was used to detect the mutation frequencies of intergenic targeting site. The ttCas12a copy number was determined by q-PCR in each T1 parental line plants and calculated by the 2−∆∆CT method. Actin7 was used as an internal reference. Supplementary Figure S4. Hygromycin-resistant phenotypes in the Cas12a/ttCas12a T2 individual candidate parental lines. A, B, Hygromycin resistance of Cas12a (A) and ttCas12a (B) T2 candidate parental lines. Labeled plant lines are the parental lines selected for sequential transformation. Supplementary Figure S5. Confirmation of precise GT events by sequencing. A, B, Sequence confirmation of GFP-KI at the EMB2410 locus (A) and amino acid substitutions at the ALS locus (B). PCR of the EMB2410 and ALS target loci was performed using full-length primers. The amplified PCR products were sequenced to confirm the precise GFP-KI and ALS S653I, G654E substitutions at the EMB2410 and ALS target loci, respectively. Lowercase letters indicate the 3' UTR of EMB2410, black squares indicate the terminator, and bold letters indicate the crRNA sequence. Supplementary Figure S6. Original uncropped gel pictures.