Home: Cell Press€¦ · Web viewThe transformed Agrobacterium EHA105 cells were collected and resuspended in liquid AAM-AS medium (OD600=0.1) containing 100μM acetosyringone. After

Plant genome editing using FnCpf1 and LbCpf1 nucleases at redefined and

altered PAM sites

Zhaohui Zhong1 Ɨ, Yingxiao Zhang2 Ɨ, Qi You3,4 Ɨ, Xu Tang1, Qiurong Ren1, Shishi Liu1, Lijia Yang1, Yan Wang1, Xiaopei Liu1, Binglin Liu1, Tao Zhang3,4, Xuelian Zheng1, Ysa Le2, Yong Zhang1*, Yiping Qi2,5*

1Department of Biotechnology, School of Life Sciences and Technology, Center for Informational Biology, University of Electronic Science and Technology of China, Chengdu 610054, China; 2Department of Plant Science and Landscape Architecture, University of Maryland, College Park, Maryland 20742, USA; 3Jiangsu Key Laboratory of Crop Genetics and Physiology, Co-Innovation Center for Modern Production Technology of Grain Crops, Key Laboratory of Plant Functional Genomics of the Ministry of Education, Yangzhou University, Yangzhou 225009, China; 4Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China, Yangzhou University, Yangzhou 225009, China; 5Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, Maryland 20850.

Ɨ These authors contributed equally to this work.*Corresponding authors: [email protected]; [email protected]

SUPPLEMENTAL INFORMATION

Supplemental Materials and Methods Supplemental Table 1. All 85 T-DNA vectors used in this study Supplemental Table 2. New Gateway-compatible Cpf1 expression vectors generated in this

study Supplemental Table 3. Oligos used in this study Supplemental Figure 1. CRISPR-Cpf1 expression system in this study Supplemental Figure 2. Mutations profiles at additional TTV and TTTV PAM sites targeted

by FnCpf1 Supplemental Figure 3. Genotyping T0 mutant lines by FnCpf1 at TTTV PAM sites with

Sanger sequencing Supplemental Figure 4. Genotyping T0 mutant lines by FnCpf1 at TTV PAM sites with

Sanger sequencing Supplemental Figure 5. RFLP analysis for editing frequencies at VTTV and VTTTV PAM

sites Supplemental Figure 6. Mutations profiles at altered PAM sites targeted by LbCpf1 variants Supplemental Figure 7. Mutations profiles at altered PAM sites targeted by FnCpf1 variants Supplemental Figure 8. Genotyping T0 mutant lines edited by Cpf1 variants with Sanger

sequencing at six sites

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mailto:[email protected]

mailto:[email protected]

SUPPLEMENTAL MATERIALS AND METHODS

Vector construction

All vectors used in this study were constructed based on a three-way Gateway cloning system.

Enzyme Esp3I (BsmBI) and Gateway™ LR Clonase™ II Enzyme Mix were from Thermo Fisher

Scientific (Waltham, MA, USA). Plasmid was isolated using IBI scientific Hi-Speed Mini Plasmid

Kit (Dubuque, IA, USA). Gel extraction was conducted using QIAquick Gel Extraction Kit

(Hilden, Germany). Oligonucleotides and gBlocks were synthesized by Integrated DNA

Technologies (Coralville, IA, USA). All other reagents used in this study were from New England

Biolabs (Ipswich, MA, USA).

The attL1-attR5 entry vectors used in this study for Cpf1 expression included pYPQ239,

pYPQ230-RR, pYPQ230-RVR, pYPQ239-RR and pYPQ239-RVR. To generate pYPQ239 for

the expression of rice codon-optimized FnCpf1, three gBlocks (FnCpf1-gBlock-1, FnCpf1-

gBlock-2, FnCpf1-gBlock-3) were synthesized and sequentially cloned into pYPQ230 (LbCpf1,

Addgene Plasmid #86210, Tang et al., 2017) at NcoI-SpeI, NcoI-BsaI and BsaI-MfeI sites. The

final vector was sequenced using primers M13-F1, M13-R1 and 239-seq1 to 239-seq7

(Supplementary Table 2). To generate LbCpf1 entry clone with RR mutations, two PCR

fragments were amplified from pYPQ230 with primers 230-RR-F1 and 230-RR-R1, 230-RR-F2

and 230-RR-R2 (Supplementary Table 2). These two fragments were co-transformed into E.coli

strain DH5α. Overlap sequences of the two PCR fragments allowed homologous recombination

to occur in E.coli and form the circular vector with LbCpf1 harboring RR mutations. The final

vector pYPQ230-RR was confirmed by Sanger sequencing. LbCpf1 entry clone with RVR

mutations (pYOQ230-RVR), as well as FnCpf1 entry clone with RR (pYPQ239-RR) and RVR

(pYPQ239-RVR) mutations, were generated using the same method, with primers listed in

Supplementary Table 2.

The attL5-attL2 entry vectors used in this study for crRNA expression were pYPQ141-ZmUbi-

RZ-Lb (Addgene Plasmid #86197, Tang et al., 2017) and pYPQ141-ZmUbi-RZ-Fn. To generate

pYPQ141-ZmUbi-RZ-Fn for Fn-crRNA expression, two DNA oligonucleotides (Fn-crRNA-F and

Fn-crRNA-R) were synthesized and phosphorylated using T4 Polynucleotide Kinase (T4 PNK).

The reaction was incubated at 37℃ for 30 min and then moved to boiling water. The reaction

was left to cool down to room temperature for annealing. At the same time, vector pYPQ141-

ZmUbi-RZ-Lb was digested with BamHI and SacI, followed by gel purification using QIAquick

Gel Extraction Kit. The digested vector was then ligated with phosphorylated and annealed

oligonucleotides to generate pYPQ141-ZmUbi-RZ-Fn. The final vector was sequencing

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confirmed with primer Ubi-intron-F1 and M13-R1 (Supplementary Table 2). To generate

individual crRNA expression vectors, crRNA was synthesized as duplexed oligonucleotides.

Oligos were phosphorylated, annealed and cloned into Esp3I (BsmBI) linearized pYPQ141-

ZmUbi-RZ-Lb or pYPQ141-ZmUbi-RZ-Fn using the following recipes. Plasmids were sequenced

using primer Ubi-intron-F1 to confirm the insertion of crRNA.

Oligonucleotides phosphorylation

Component VolumecrRNA forward oligo (100 µM) 1 µL crRNA reverse oligo (100 µM) 1 µL T4 PNK Reaction Buffer (10X) 1 µL ATP (10 mM) 1 µL T4 PNK (10U/µL) 0.5 µL (5U)Nuclease-free Water 5.5 µLTotal 10 µL

Reactions were incubated at 37℃ for 30 min and moved to boiling water. Reactions were left to cool down to room temperature.

Empty crRNA expression vector digestion

Component VolumepYPQ141-ZmUbi-RZ-Lb or pYPQ141-ZmUbi-RZ-Fn 20 µL (2 µg)

Buffer Tango (10X) 5 µL DTT (10 mM) 5 µL Esp3I (BsmBI) (10U/µL) 2 µL (20U)Nuclease-free Water 18 µLTotal 50 µL

Reactions were incubated at 37℃ overnight followed by gel purification using QIAquick Gel Extraction Kit.

crRNA expression vector ligation

Component VolumeEsp3I (BsmBI) linearized pYPQ141-ZmUbi-RZ-Lb or pYPQ141-ZmUbi-RZ-Fn 2 µL (50 ng)

Diluted annealed oligos (1:200 dilution) 2 µL T4 DNA Ligase Buffer (10X) 2 µL T4 DNA Ligase (400U/µL) 1 µL (400U)Nuclease-free Water 13 µLTotal 20 µL

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Reactions were incubated at room temperature for two hours followed by E.coli transformation.

The Cpf1 expression entry vector, crRNA expression entry vector and the destination vector

pYPQ203 (pMDC32-Ubi1, Addgene Plasmid #86207, Tang et al., 2017) were assembled using

a three-way Gateway cloning system. LR reactions were set up as following:

Component VolumeCpf1 expression entry vector 1.5 µL (150 ng)crRNA expression entry vector 1 µL (100 ng)Destination vector pYPQ203 2 µL (200 ng)LR Clonase II 1 µlTotal 5.5 µl

Reactions were incubated at room temperature for one hour or overnight and transformed into

E.coli to generate the final T-DNA vectors. These vectors were confirmed by EcoRI digestion.

Plant material and growth condition

The Japonica cultivar Nipponbare was used in this study. For protoplast preparation, the

sterilized seeds were placed on the 1/2 MS solid medium for 11 days in dark chamber at 28℃.

For rice stable transformation, the sterilized seeds were placed on the N6-D solid medium to

induce calli for 7 days under light at 32℃.

Rice protoplast preparation and transformation

The rice protoplast isolation and transformation were performed based on the protocol we

published before (Tang et al., 2016). Briefly, the healthy leaves were cut into 0.5-1.0 mm strips

and transferred into the enzyme solution followed by vacuum-infiltration for 30 minutes. The

mixture was incubated at 25℃ for 8 hours in the dark. The enzyme solution mixture was then

filtered by 40μm cell strainer. After washing two times by W5 washing buffer, the number of

protoplasts for each sample was counted using a microscope. The final protoplast concentration

was adjusted to 2x106 per milliliter.

For protoplast transformation, 40μL plasmid DNA (total 30μg) was used for 200μL protoplast.

Briefly, they were gently mixed with 240μL PEG transformation buffer. After incubation for 30

minutes at room temperature in dark, the reactions were stopped by adding 900μL W5 washing

buffer. The protoplast were collected by low speed centrifugation and transferred into 12-well

culture plate for further incubation in WI solution at 32℃ in dark for 2 days. The protoplasts

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were collected after 2 days, and DNA was extracted using the CTAB method (Murray and

Thompson, 1980).

RFLP analysis for mutation frequency

PCR was conducted with the Tsingke Golden Mix kit (Tsingke) with specific primers. The PCR

products were digested with the restriction enzyme of choice overnight. The digested products

were analyzed with electrophoresis in 1% TAE agrose gels. The mutation frequency was

quantified by ImageJ software.

The NGS sample preparation, detection and analysis

Next-generation sequencing (NGS) of PCR amplicons was also used for detection and

quantification of mutations for the samples. With the protoplast DNA as template, the flanking

sequence of each target site was PCR-amplified using KOD-PLUS–NEO (Toyobo) and the

barcodes were added at each end of the primers. The PCR products were run on a 1% agrose

gel for checking purity and the concentration of DNA was estimated by comparing with the DNA

markers. Then the PCR product were sent to the Novogene for sequencing using Illumina Hiseq

2500 platform. More than 50000 reads were generated for each sample and the clean data

were mapped by BWA (Li and Durbin, 2010). The data processing and further analysis were

carried out using python and R.

Rice stable transformation and genotyping of T0 lines

The Agrobacterium-mediated rice transformation was performed as described in the published

protocols (Hiei et al., 1994). Briefly, the calli were induced in the growth chamber for 7 days at

32℃ under light. The binary vectors were transferred into Agrobacterium tumefaciens strain

EHA105. The transformed Agrobacterium EHA105 cells were collected and resuspended in

liquid AAM-AS medium (OD600=0.1) containing 100μM acetosyringone. After co-cultured the

agrobacterium and the calli for 3 days, the calli were washed by sterilized water and transferred

to N6-S medium contained 400mg/L Carbenicillin and 50mg/L Hygromycin for 2 weeks. Then

calli were transferred to REIII medium with 400mg/L Carbenicillin and 50mg/L Hygromycin

cultured for 2 weeks and he resistant calli were moved to fresh REIII for every 2 weeks until

obtaining regenerated plants. DNA from T0 plants was extracted with the CTAB method (Murray

and Thompson, 1980). Mutations in T0 plants were screened by the RFLP assay and further

genotyped by Sanger sequencing.

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Supplemental References

Hiei, Y., Ohta, S., Komari, T., and Kumashiro, T. (1994). Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J 6:271-282.

Li, H., and Durbin, R. (2010). Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics 26:589-595.

Murray, M.G., and Thompson, W.F. (1980). Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8:4321-4325.

Tang, X., Lowder, L.G., Zhang, T., Malzahn, A.A., Zheng, X., Voytas, D.F., Zhong, Z., Chen, Y., Ren, Q., Li, Q., et al. (2017). A CRISPR-Cpf1 system for efficient genome editing and transcriptional repression in plants. Nat Plants 3: 17103.

Tang, X., Zheng, X., Qi, Y., Zhang, D., Cheng, Y., Tang, A., Voytas, D.F., and Zhang, Y. (2016). A single transcript CRISPR-Cas9 system for efficient genome editing in plants. Mol Plant 9:1088-1091.

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Supplemental Table 1. All 85 T-DNA vectors used in this study

Name of T-DNA vectors LR # crRNA sequence PAMpYPQ203-FnCpf1-ZmUbi-RZ-Fn-A1 450 GAGTGAAATCTCTTGTCTTAAGG TTTG

pYPQ203-FnCpf1-ZmUbi-RZ-Fn-A2 451 TTCAAAACCCTTAGAGATATCTA TTTG

pYPQ203-FnCpf1-ZmUbi-RZ-Fn-A3 452 CTACTGTTGCAAGTGCTCACCCA TTTC

pYPQ203-FnCpf1-ZmUbi-RZ-Fn-A4 453 CAGAAAGAGAAGGAGGCACAGAT TTTC

pYPQ203-FnCpf1-ZmUbi-RZ-Fn-A5 454 TGCTTCCTGCAATGCCGGTAGAC TTTG

pYPQ203-FnCpf1-ZmUbi-RZ-Fn-A6 455 TAAGCAGCTGGCTGAGGGTGCAT TTTG

pYPQ203-FnCpf1-ZmUbi-RZ-Fn-L1 456 AGGAATAAAGGAAAAAGATTCCG CTTA

pYPQ203-FnCpf1-ZmUbi-RZ-Fn-L2 457 GAAAGTCCTGGCAAACAACCTGC CTTG

pYPQ203-FnCpf1-ZmUbi-RZ-Fn-L3 458 CTGGGCTCGGCGTCCTCGACGTC GTTG

pYPQ203-FnCpf1-ZmUbi-RZ-Fn-L4 459 CATCACACAGCCGGAAGGTACCC CTTG

pYPQ203-FnCpf1-ZmUbi-RZ-Fn-L5 460 CTCAAGGTGAGCGCCCCGCGGCG GTTG

pYPQ203-FnCpf1-ZmUbi-RZ-Fn-L6 461 ACTTCCTTGAAGGAGCTCAGCCC CTTC

pYPQ203-FnCpf1-ZmUbi-RZ-Fn-L7 462 AAACTGAATCTCCTGCTCCTCTG CTTC

pYPQ203-FnCpf1-ZmUbi-RZ-Fn-L8 463 CCTCTCTCTCCTGTGCTTGCCTC TTTG

pYPQ203-FnCpf1-ZmUbi-RZ-Fn-L9 464 AAGAAGGGTTATGGCCAATGCTT TTTG

pYPQ203-FnCpf1-ZmUbi-RZ-Fn-L10 465 CAATCAAGGGCACCATGGCAGCA GTTG

pYPQ203-FnCpf1-ZmUbi-RZ-Fn-L11 466 GATTGAATTTATTTCACCCTGCA CTTC

pYPQ203-FnCpf1-ZmUbi-RZ-Fn-L12 467 ATCGGAAGAACTCCTGATCCATT ATTC

pYPQ203-LbCpf1-RR-N1 541 ACGGAATCTTTTTCCTTTATTCC TCCG

pYPQ203-LbCpf1-RR-N2 542 GCATGGCATTTCTACCTTATCGA TCCC

pYPQ203-LbCpf1-RR-N3 543 TTTATTCCTTAAGACAAGAGATT TTCC

pYPQ203-LbCpf1-RR-N4 544 TACTTCCCGCATGGCATTTCTAC TTCA

pYPQ203-LbCpf1-RVR-N5 545 AAACTCTTTCATCGATAAGGTAG TATC

pYPQ203-LbCpf1-RVR-N6 546 AAGTGATGTCTGAGGATTAACAC TATG

pYPQ203-LbCpf1-RR-N7 547 TAATTACAGTCATGTGTTAGTAA CCCC

pYPQ203-LbCpf1-RR-N8 548 GTTTTGTAAGCAGCTGGCTGAGG TCCG

pYPQ203-LbCpf1-RR-N9 549 TTGATGGTTTAGTGAGAGATCAT TCCA

pYPQ203-LbCpf1-RR-N10 550 TGCAATGCCGGTAGACACCTCCT TTCC

pYPQ203-LbCpf1-RVR-N12 552 ATCTCTCACTAAACCATCAATGG TATG

pYPQ203-LbCpf1-RR-N13 553 GCCATGCTCGGGGAGGTGTCCCT CCCC

pYPQ203-LbCpf1-RR-N14 554 GAAAGAGAAGGAGGCACAGATCT TCCA

pYPQ203-LbCpf1-RR-N15 555 TGCAGATCAAAATTGTGTATCTG TCCC

pYPQ203-LbCpf1-RR-N16 556 AGAAAGAGAAGGAGGCACAGATC TTCC

pYPQ203-LbCpf1-RR-N17 557 TGTCTTTGCTACTGTTGCAAGTG TTCA

pYPQ203-LbCpf1-RVR-N18 558 TTTTGATCCAATATAGTTTGCTC TATG

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pYPQ203-LbCpf1-RVR-N19 559 TGCATTTCATGTCTTTGCTACTG TATC

pYPQ203-LbCpf1-RR-N20 560 TCAGAACCTGCGAGCAAACTATA CCCC

pYPQ203-FnCpf1-RR-N1 561 ACGGAATCTTTTTCCTTTATTCC TCCG

pYPQ203-FnCpf1-RR-N2 562 GCATGGCATTTCTACCTTATCGA TCCC

pYPQ203-FnCpf1-RR-N3 563 TTTATTCCTTAAGACAAGAGATT TTCC

pYPQ203-FnCpf1-RR-N4 564 TACTTCCCGCATGGCATTTCTAC TTCA

pYPQ203-FnCpf1-RVR-N5 565 AAACTCTTTCATCGATAAGGTAG TATC

pYPQ203-FnCpf1-RVR-N6 566 AAGTGATGTCTGAGGATTAACAC TATG

pYPQ203-FnCpf1-RR-N7 567 TAATTACAGTCATGTGTTAGTAA CCCC

pYPQ203-FnCpf1-RR-N8 568 GTTTTGTAAGCAGCTGGCTGAGG TCCG

pYPQ203-FnCpf1-RR-N9 569 TTGATGGTTTAGTGAGAGATCAT TCCA

pYPQ203-FnCpf1-RR-N10 570 TGCAATGCCGGTAGACACCTCCT TTCC

pYPQ203-FnCpf1-RVR-N12 572 ATCTCTCACTAAACCATCAATGG TATG

pYPQ203-FnCpf1-RR-N13 573 GCCATGCTCGGGGAGGTGTCCCT CCCC

pYPQ203-FnCpf1-RR-N14 574 GAAAGAGAAGGAGGCACAGATCT TCCA

pYPQ203-FnCpf1-RR-N15 575 TGCAGATCAAAATTGTGTATCTG TCCC

pYPQ203-FnCpf1-RR-N16 576 AGAAAGAGAAGGAGGCACAGATC TTCC

pYPQ203-FnCpf1-RR-N17 577 TGTCTTTGCTACTGTTGCAAGTG TTCA

pYPQ203-FnCpf1-RVR-N18 578 TTTTGATCCAATATAGTTTGCTC TATG

pYPQ203-FnCpf1-RVR-N19 579 TGCATTTCATGTCTTTGCTACTG TATC

pYPQ203-FnCpf1-RR-N20 580 TCAGAACCTGCGAGCAAACTATA CCCC

pLR458-MS1 458-MS1

GAGGGCTCGGCGTCCTCGACGTC GTTG

pLR458-MS2 458-MS2

CTGGATTCGGCGTCCTCGACGTC GTTG

pLR458-MS3 458-MS3

CTGGGCTCCCCGTCCTCGACGTC GTTG

pLR458-MS4 458-MS4

CTGGGCTCGGCGAGCTCGACGTC GTTG

pLR458-MS5 458-MS5

CTGGGCTCGGCGTCCTATACGTC GTTG

pLR458-MS6 458-MS6

CTGGGCTCGGCGTCCTCGACCAC GTTG

pLR464-MS1 464-MS1

TTGAAGGGTTATGGCCAATGCTT TTTG

pLR464-MS2 464-MS2

AAGATCGGTTATGGCCAATGCTT TTTG

pLR464-MS3 464-MS3

AAGAAGGGAAATGGCCAATGCTT TTTG

pLR464-MS4 464-MS4

AAGAAGGGTTATTACCAATGCTT TTTG

pLR464-MS5 464-MS5

AAGAAGGGTTATGGCCTCTGCTT TTTG

pLR464-MS6 464-MS6

AAGAAGGGTTATGGCCAATGGAT TTTG

pYPQ203-FnCpf1-ZmUbi-RZ-Fn-AA1 669 TCAGGTTCAGAATGCTGGTCCCC ATTTA/ATTA

pYPQ203-FnCpf1-ZmUbi-RZ-Fn-AA2 670 CCGGTGAAAAGGACCTTGTCCCA ATTTA/ATTA

pYPQ203-FnCpf1-ZmUbi-RZ-Fn-AC1 671 TGGGGCCTTGCAAGGTCACCTCC ATTTC/ATTC

pYPQ203-FnCpf1-ZmUbi-RZ-Fn-AC2 672 GTCTCCCTGCAAACCACCGCGCA ATTTC/ATTC

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pYPQ203-FnCpf1-ZmUbi-RZ-Fn-AG1 673 CCGGCAGCTAATAGGGATCTAAA ATTTG/ATTG

pYPQ203-FnCpf1-ZmUbi-RZ-Fn-AG2 674 GGGCATGGAGACAGGAGACATAG ATTTG/ATTG

pYPQ203-FnCpf1-ZmUbi-RZ-Fn-CA1 675 TACGTGGAAACAATGACAGTTCA CTTTA/CTTA

pYPQ203-FnCpf1-ZmUbi-RZ-Fn-CA1 676 ACCCTGTGTGAATGGTCAGTAAG CTTTA/CTTA

pYPQ203-FnCpf1-ZmUbi-RZ-Fn-CC1 677 TCCTGAGGAGCAAGAGCCATCAC CTTTC/CTTC

pYPQ203-FnCpf1-ZmUbi-RZ-Fn-CC2 678 TGTATCTCCGACACCCGGATCAC CTTTC/CTTC

pYPQ203-FnCpf1-ZmUbi-RZ-Fn-CG1 679 GATGATGCATCAGGTACTAGAAC CTTTG/CTTG

pYPQ203-FnCpf1-ZmUbi-RZ-Fn-CG2 680 GCACCATATGCTTGCTGATCAAA CTTTG/CTTG

pYPQ203-FnCpf1-ZmUbi-RZ-Fn-GA1 681 TAATATTGGATCCACCGATTTAA GTTTA/GTTA

pYPQ203-FnCpf1-ZmUbi-RZ-Fn-GA2 682 GTATTTCTGAATTTCATGCCCAA GTTTA/GTTA

pYPQ203-FnCpf1-ZmUbi-RZ-Fn-GC1 683 TTCACAATCTTATAGTCACTGAA GTTTC/GTTC

pYPQ203-FnCpf1-ZmUbi-RZ-Fn-GC2 684 TGAATGCTCCGGGAGCCGTAGAA GTTTC/GTTC

pYPQ203-FnCpf1-ZmUbi-RZ-Fn-GG1 685 AGCATATGGTTGTAACTTCAGAA GTTTG/GTTG

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Supplemental Table 2. New Gateway-compatible Cpf1 expression vectors generated in this study

Addgene ID Plasmid Gateway recombination sites Annotation

108859 pYPQ239 attL1-attR5 FnCpf1 Gateway entry plasmid

108860 pYPQ230-RR attL1-attR5 LbCpf1 Gateway entry plasmid with G548R and K611R mutations

108861 pYPQ230-RVR attL1-attR5 LbCpf1 Gateway entry plasmid with G548R, K554V and Y558R mutations

108862 pYPQ239-RR attL1-attR5 FnCpf1 Gateway entry plasmid with N623R and K687R mutations

108863 pYPQ239-RVR attL1-attR5 FnCpf1 Gateway entry plasmid with N623R, K629R and N633R mutations

108864 pYPQ141-ZmUbi-RZ-Fn attL5-attL2FnCpf1 Gateway crRNA entry plasmid using Zea mays Ubi promoter and double ribozyme processing

Supplemental Table 3. Oligos used in this study.

Name Sequence 5'-3' DescriptionOsPDS-HTS-F1

gcATGgatactggctgcct forward oligo to amplify the LbCpf1-OsPDS-TCCG-crRNA01 target site

OsPDS-HTS-R1

GGAGGTCTTGGAAAGTCCTGG reverse oligo to amplify the LbCpf1-OsPDS-TCCG-crRNA01 target site

OsPDS-HTS-F1

gcATGgatactggctgcct forward oligo to amplify the LbCpf1-OsPDS-TTCC-crRNA01 target site

OsPDS-HTS-R1

GGAGGTCTTGGAAAGTCCTGG reverse oligo to amplify the LbCpf1-OsPDS-TTCC-crRNA01 target site

OsPDS-HTS-F2

GGAAGGATGAAGATGGAGATTG forward oligo to amplify the LbCpf1-OsPDS-TATC-crRNA01 target site

OsPDS-HTS-R2

gctcatgatatttatgtgacgttaa reverse oligo to amplify the LbCpf1-OsPDS-TATC-crRNA01 target site

LR547-LR567-HTS-F

TTCAGCCGGTTTGATTTTCCTG forward oligo to amplify the LbCpf1-OsPDS-CCCC-crRNA01 target site

LR547-LR567-HTS-R

GGCCAAGTTAGCATTTCATTGTTTCT reverse oligo to amplify the LbCpf1-OsPDS-CCCC-crRNA01 target site

LR549-LR569-F

gtgtaagacgtagctaacatattttctt forward oligo to amplify the LbCpf1-OsROC5-TCCG-crRNA01 target site

LR549-LR569-R

tgacaatactattaactgacttactgcc reverse oligo to amplify the LbCpf1-OsROC5-TCCG-crRNA01 target site

OsROC5-HTS-F1

AGgtttgggctaatgtcctcc forward oligo to amplify the LbCpf1-OsROC5-TTCC-crRNA01 target site

OsROC5-HTS-R1

CACCTGTAGCTCAGCCTTCAT reverse oligo to amplify the LbCpf1-OsROC5-TTCC-crRNA01 target site

LR549-LR569-F

gtgtaagacgtagctaacatattttctt forward oligo to amplify the LbCpf1-OsROC5-TATG-crRNA01 target site

LR549-LR569-R

tgacaatactattaactgacttactgcc reverse oligo to amplify the LbCpf1-OsROC5-TATG-crRNA01 target site

LR553-LR573-F

tcgtgtgccttagtcttcttctc forward oligo to amplify the LbCpf1-OsROC5-CCCC-crRNA01 target site

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LR553-LR573-R

TAGGAACTTGGTGGCGAGG reverse oligo to amplify the LbCpf1-OsROC5-CCCC-crRNA01 target site

OsDEP1-HTS-F2

TGGTACAAAACATGACCCACT forward oligo to amplify the FnCpf1-OsDEP1-TCCA-crRNA01 target site

OsDEP1-HTS-R2

gggactagaggcactttcaga reverse oligo to amplify the FnCpf1-OsDEP1-TCCA-crRNA01 target site

OsDEP1-HTS-F2

TGGTACAAAACATGACCCACT forward oligo to amplify the FnCpf1-OsDEP1-TTCC-crRNA01 target site

OsDEP1-HTS-R2

gggactagaggcactttcaga reverse oligo to amplify the FnCpf1-OsDEP1-TTCC-crRNA01 target site

OsDEP1-HTS-F2

TGGTACAAAACATGACCCACT forward oligo to amplify the FnCpf1-OsDEP1-TATG-crRNA01 target site

OsDEP1-HTS-R2

gggactagaggcactttcaga reverse oligo to amplify the FnCpf1-OsDEP1-TATG-crRNA01 target site

OsDEP1-HTS-F2

TGGTACAAAACATGACCCACT forward oligo to amplify the FnCpf1-OsDEP1-CCCC-crRNA01 target site

OsDEP1-HTS-R2

gggactagaggcactttcaga reverse oligo to amplify the FnCpf1-OsDEP1-CCCC-crRNA01 target site

OsPDS-HTS-F1

gcATGgatactggctgcct forward oligo to amplify the OsPDS-TTTV-crRNA01 target site

OsPDS-HTS-R1

GGAGGTCTTGGAAAGTCCTGG reverse oligo to amplify the OsPDS-TTTV-crRNA01 target site

OsDEP1-HTS-F2

TGGTACAAAACATGACCCACT forward oligo to amplify the OsDEP1-TTTV-crRNA02 target site

OsDEP1-HTS-R2

gggactagaggcactttcaga reverse oligo to amplify the OsDEP1-TTTV-crRNA02 target site

456-F gatactggctgcctgtcatcta forward oligo to amplify the OsPDS-TTV-crRNA01 target site

456-R ttttctagtggaggtcttggaa reverse oligo to amplify the OsPDS-TTV-crRNA01 target site

457-F tagtcagcatgtgagctttgga forward oligo to amplify the OsPDS-TTV-crRNA02 target site

457-R atatcatacctgctccagcaat reverse oligo to amplify the OsPDS-TTV-crRNA02 target site

458-F tattttgttgacaggacaacgc forward oligo to amplify the OsROC5-TTV-crRNA01 target site

458-R gttggcacagaggagaaggatg reverse oligo to amplify the OsROC5-TTV-crRNA01 target site

460-F tctgagttggccgttggcgtgc forward oligo to amplify the OsDEP1-TTV-crRNA01 target site

460-R ccgtactcggccgcttgggttt reverse oligo to amplify the OsDEP1-TTV-crRNA01 target site

464-F tatgcctggcatgagcagagcc forward oligo to amplify the OsEPFL9-TTTV-crRNA01 target site

464-R aggagaacagaagaaaagacaaga reverse oligo to amplify the OsEPFL9-TTTV-crRNA01 target site

465-F gttgcttctctaatccaggcacac forward oligo to amplify the OsEPFL9-TTV-crRNA02 target site

465-R tgttcttcaggtaatgtttggtg reverse oligo to amplify the OsEPFL9-TTV-crRNA02 target site

AA-TTN-Eco47I-F1

TCATGGTTCTGATCTGGCATGT forward oligo to amplify the ATTA-crRNA01 target site

AA-TTN-Eco47I-R1

GATCAGATGGTGTTTGGCCC reverse oligo to amplify the ATTA-crRNA01 target site

AA-TTTN-Eco47I-F

GCAATCAATGTCTACCGCGT forward oligo to amplify the ATTTA-crRNA01 target site

AA-TTTN- AGGCTGCTGGTATTGTTGGA reverse oligo to amplify the ATTTA-crRNA01

11

Eco47I-R target siteAA-TTN-PsyI-F

GCCACCGCTAAGTTCCTCG forward oligo to amplify the ATTA-crRNA02 target site

AA-TTN-PsyI-R

GTCGAAGATGCAGGGGCAC reverse oligo to amplify the ATTA-crRNA02 target site

AA-TTTN-PsyI-F

TTCTGAAGCCCTCCGAGTTT forward oligo to amplify the ATTTA-crRNA02 target site

AA-TTTN-PsyI-R

TAATGATGCGCCCTACACGA reverse oligo to amplify the ATTTA-crRNA02 target site

AC-TTN-NmuCI-F

TTCCCTCACCGGCTTGAATC forward oligo to amplify the ATTC-crRNA01 target site

AC-TTN-NmuCI-R

GTACTGTCCTGTGCCGCTAA reverse oligo to amplify the ATTC-crRNA01 target site

AC-TTTN-NmuCI-F

TTCTTCAGGCATTGCCCAGT forward oligo to amplify the ATTTC-crRNA01 target site

AC-TTTN-NmuCI-R

GCAGGAGCCCAGCTGTATAA reverse oligo to amplify the ATTTC-crRNA01 target site

AC-TTTN-SsiI-F

GAAATTCCCGTGCGTTTCAGG forward oligo to amplify the ATTC-crRNA02 target site

AC-TTTN-SsiI-R

CCCAACGCACCAACATTGTC reverse oligo to amplify the ATTC-crRNA02 target site

AC-TTN-SsiI-F

TGCTTGGCATCGTTGCTCTAT forward oligo to amplify the ATTTC-crRNA02 target site

AC-TTN-SsiI-R

CGCCTCATGCTCCATCACT reverse oligo to amplify the ATTTC-crRNA02 target site

AG-TTN-Sau3AI-F

CCCATGGAGAGTCAGGTGC forward oligo to amplify the ATTG-crRNA01 target site

AG-TTN-Sau3AI-R

GAAGATGGACGGCAAGGAGAT reverse oligo to amplify the ATTG-crRNA01 target site

AG-TTTN-Sau3AI-F1

CCGCTGCTCCGGGAC forward oligo to amplify the ATTTG-crRNA01 target site

AG-TTTN-Sau3AI-R1

CAAGCCCGACATACCAGTCA reverse oligo to amplify the ATTTG-crRNA01 target site

AG-TTN-Chr11-F

CGCCGTGTACAGGTTGTGTA forward oligo to amplify the ATTG-crRNA02 target site

AG-TTN-Chr11-R

TCTAAGCTTCCCTTTGGAGCC reverse oligo to amplify the ATTG-crRNA02 target site

AG-TTTN-Chr11-F

GAAAAAATTTTAAAACATAAGTCACGAG forward oligo to amplify the ATTTG-crRNA02 target site

AG-TTTN-Chr11-R

AAGTTCAGGGATGTTCAGAGCA reverse oligo to amplify the ATTTG-crRNA02 target site

CA-TTN-TaaI-F

GAAAGCAGAGGAGAGCTGCG forward oligo to amplify the CTTA-crRNA01 target site

CA-TTN-TaaI-R

GAAGGGACTGATTAGTGCCAAA reverse oligo to amplify the CTTA-crRNA01 target site

CA-TTTN-TaaI-F

GAAAATGCAAAGTTGAATTACCAGT forward oligo to amplify the CTTTA-crRNA01 target site

CA-TTTN-TaaI-R

ACAGTTCAATTCCTTGAGATTCTGT reverse oligo to amplify the CTTTA-crRNA01 target site

CA-TTN-36070-F

CCTGTTGTTCGTCGACCTCT forward oligo to amplify the CTTA-crRNA02 target site

CA-TTN-36070-R

TAGGTCATATAGCAGCGTGCG reverse oligo to amplify the CTTA-crRNA02 target site

CA-TTTN-40824-F

GCTGTATGTTTGTATGGTCGCC forward oligo to amplify the CTTTA-crRNA02 target site

CA-TTTN-40824-R

AACGAATTCAACACGGGATGG reverse oligo to amplify the CTTTA-crRNA02 target site

12

CC-TTN-BccI-F

AGCTGGTGCCACTGTTGAATA forward oligo to amplify the CTTC-crRNA01 target site

CC-TTN-BccI-R

CAGGGTAGCATGACGAGGTTA reverse oligo to amplify the CTTC-crRNA01 target site

CC-TTTN-BccI-F

AGCTGGTGCCACTGTTGAAT forward oligo to amplify the CTTTC-crRNA01 target site

CC-TTTN-BccI-R

GCAAAGTGGTATATCAAGACAGCA reverse oligo to amplify the CTTTC-crRNA01 target site

CC-TTN-Sau3AI-F1

GCCATCCAGGTCAGTCTGTTA forward oligo to amplify the CTTC-crRNA02 target site

CC-TTN-Sau3AI-R1

GAGCAGAAGCGGAAGGAGTC reverse oligo to amplify the CTTC-crRNA02 target site

CC-TTTN-Sau3AI-F

ACATGTACTGCCTCGACTGC forward oligo to amplify the CTTTC-crRNA02 target site

CC-TTTN-Sau3AI-R1

CAGAAGCGGAAGGTGTCGAG reverse oligo to amplify the CTTTC-crRNA02 target site

CG-TTN-BfaI-F

CATCCCAAGGAGACGCATGT forward oligo to amplify the CTTG-crRNA01 target site

CG-TTN-BfaI-R

GCTTACTCGAGTCGGTGGTG reverse oligo to amplify the CTTG-crRNA01 target site

CG-TTTN-BfaI-F1

CACCCACATCGACACCCTTG forward oligo to amplify the CTTTG-crRNA01 target site

CG-TTTN-BfaI-R1

TTGGATCCTGGCCAATGCAA reverse oligo to amplify the CTTTG-crRNA01 target site

CG-TTN-Sau3AI-F

AGTAGAAGGCAGTCTTGAGGAT forward oligo to amplify the CTTG-crRNA02 target site

CG-TTN-Sau3AI-R

CGAGCTTTTGTTAAGAAGTGCCA reverse oligo to amplify the CTTG-crRNA02 target site

CG-TTTN-Sau3AI-F

ACAGAAAGCGTCAAACTTTCCTTA forward oligo to amplify the CTTTG-crRNA02 target site

CG-TTTN-Sau3AI-R

TGGCATGACTAGATTTAGTGCCC reverse oligo to amplify the CTTTG-crRNA02 target site

GA-TTN-BamHI-F

GTACACGGGAAGGAGTAACCA forward oligo to amplify the GTTA-crRNA01 target site

GA-TTN-BamHI-R

ACCTCAAGGGTGGATCCGTTA reverse oligo to amplify the GTTA-crRNA01 target site

GA-TTTN-BamHI-F

acaaatttgggtcatgacactct forward oligo to amplify the GTTTA-crRNA01 target site

GA-TTTN-BamHI-R

tgcaagcagtaccgtaccta reverse oligo to amplify the GTTTA-crRNA01 target site

GA-TTN-01020-F

TCATGATTTTCCCGTCGACCTA forward oligo to amplify the GTTA-crRNA02 target site

GA-TTN-01020-R

CTCGTGCCCAACAATCCAAG reverse oligo to amplify the GTTA-crRNA02 target site

GA-TTTN-Chr7-F

ggggaaacgtattcgtgcat forward oligo to amplify the GTTTA-crRNA02 target site

GA-TTTN-Chr7-R

AATTCATGATTTTTCCTGTCGACTT reverse oligo to amplify the GTTTA-crRNA02 target site

GC-TTN-NmuCI-F

CTCCTTCAGGGCCACTTCTA forward oligo to amplify the GTTC-crRNA01 target site

GC-TTN-NmuCI-R

TAGTTAAGCCTCCGCCTTGG reverse oligo to amplify the GTTC-crRNA01 target site

GC-TTTN-NmuCI-F

GGTTGAACCATCTTTGGGGC forward oligo to amplify the GTTTC-crRNA01 target site

GC-TTTN-NmuCI-R

GCACAGTTCATGAAGTATCCGA reverse oligo to amplify the GTTTC-crRNA01 target site

GC-TTN- TGCTGATCGAGGCAATAGGT forward oligo to amplify the GTTC-crRNA02

13

BceAI-F1 target siteGC-TTN-BceAI-R1

GCCAGACATGGTTACTGGTGA reverse oligo to amplify the GTTC-crRNA02 target site

GC-TTTN-BceAI-F

ACGGTTCTCTGTCAGCATCA forward oligo to amplify the GTTTC-crRNA02 target site

GC-TTTN-BceAI-R

TTATGCAGGGTTGCGGTGAA reverse oligo to amplify the GTTTC-crRNA02 target site

GG-TTN-Eco57I-F1

GATGATATTGATGATTCTTCTGGAG forward oligo to amplify the GTTG-crRNA01 target site

GG-TTN-Eco57I-R1

CTGGGAGACATGAAACACCG reverse oligo to amplify the GTTG-crRNA01 target site

GG-TTTN-Eco57I-F1

TGGTCTTTTGCCGTCTTTGT forward oligo to amplify the GTTTG-crRNA01 target site

GG-TTTN-Eco57I-R

AAACAGAATGAACTTGACTGGCT reverse oligo to amplify the GTTTG-crRNA01 target site

GG-TTN-23770-F

AGTTTCACTGGCCCCTTACTG forward oligo to amplify the GTTG-crRNA02 target site

GG-TTN-23770-R1

CAGTAGGCTGTTGTTGTGGC reverse oligo to amplify the GTTG-crRNA02 target site

GG-TTTN-41190-F1

CATGCAATTCAGGCAAAAGCC forward oligo to amplify the GTTTG-crRNA02 target site

GG-TTTN-41190-R1

GCACGAGTCCCTGGATTTCA reverse oligo to amplify the GTTTG-crRNA02 target site

Oligos used to amplify target sites for NGSName Sequence 5'-3' DescriptionLR450-HTS-F

CACTCActggctgcctgtcatctatgaa forward oligo to amplify the OsPDS-TTTV-crRNA01 target site

LR450-HTS-R

CAGGCGggaggtcttggaaagtcctgg reverse oligo to amplify the OsPDS-TTTV-crRNA01 target site

LR450-HTS-R1

CATGGCggaggtcttggaaagtcctgg reverse oligo to amplify the OsPDS-TTTV-crRNA01 target site

LR450-HTS-R2

CATTTTggaggtcttggaaagtcctgg reverse oligo to amplify the OsPDS-TTTV-crRNA01 target site

LR451-HTS-F

GGCTACggaaggatgaagatggagattg forward oligo to amplify the OsPDS-TTTV-crRNA02 target site

LR451-HTS-R

CTTGTAgctcatgatatttatgtgacgttaa reverse oligo to amplify the OsPDS-TTTV-crRNA02 target site

LR451-HTS-R1

AGTCAAgctcatgatatttatgtgacgttaa reverse oligo to amplify the OsPDS-TTTV-crRNA02 target site

LR451-HTS-R2

AGTTCCgctcatgatatttatgtgacgttaa reverse oligo to amplify the OsPDS-TTTV-crRNA02 target site

LR452-HTS-F

ATGTCAggcataataatctgtactactgcca forward oligo to amplify the OsDEP1-TTTV-crRNA01 target site

LR452-HTS-R

CCGTCCgaaggggtcttgcagcaact reverse oligo to amplify the OsDEP1-TTTV-crRNA01 target site

LR452-HTS-R1

GTAGAGgaaggggtcttgcagcaact reverse oligo to amplify the OsDEP1-TTTV-crRNA01 target site

LR452-HTS-R2

GTCCGCgaaggggtcttgcagcaact reverse oligo to amplify the OsDEP1-TTTV-crRNA01 target site

LR453-HTS-F

CAAAAGtggtacaaaacatgacccact forward oligo to amplify the OsDEP1-TTTV-crRNA02 target site

LR453-HTS-R

CAACTAgggactagaggcactttcaga reverse oligo to amplify the OsDEP1-TTTV-crRNA02 target site

LR453-HTS-R1

CACCGGgggactagaggcactttcaga reverse oligo to amplify the OsDEP1-TTTV-crRNA02 target site

LR453-HTS-R2

CACGATgggactagaggcactttcaga reverse oligo to amplify the OsDEP1-TTTV-crRNA02 target site

14

LR454-HTS-F

GTGGCCaggtttgggctaatgtcctcc forward oligo to amplify the OsROC5-TTTV-crRNA01 target site

LR454-HTS-R

GTTTCGcacctgtagctcagccttcat reverse oligo to amplify the OsROC5-TTTV-crRNA01 target site

LR454-HTS-R1

CGTACGcacctgtagctcagccttcat reverse oligo to amplify the OsROC5-TTTV-crRNA01 target site

LR454-HTS-R2

GAGTGGcacctgtagctcagccttcat reverse oligo to amplify the OsROC5-TTTV-crRNA01 target site

LR455-HTS-F

GGTAGCgctgctggtgagtgctgat forward oligo to amplify the OsROC5-TTTV-crRNA02 target site

LR455-HTS-R

ACTGATacccattgggagtgtcttgc reverse oligo to amplify the OsROC5-TTTV-crRNA02 target site

LR455-HTS-R1

ATGAGCacccattgggagtgtcttgc reverse oligo to amplify the OsROC5-TTTV-crRNA02 target site

LR455-HTS-R2

ATTCCTacccattgggagtgtcttgc reverse oligo to amplify the OsROC5-TTTV-crRNA02 target site

HTS-456-F CGATGTgatactggctgcctgtcatcta forward oligo to amplify the OsPDS-TTV-crRNA01 target site

HTS-456-R TCATTCttttctagtggaggtcttggaa reverse oligo to amplify the OsPDS-TTV-crRNA01 target site

HTS-456-R1

TATAATttttctagtggaggtcttggaa reverse oligo to amplify the OsPDS-TTV-crRNA01 target site

HTS-456-R2

TACAGCttttctagtggaggtcttggaa reverse oligo to amplify the OsPDS-TTV-crRNA01 target site

HTS-457-F ATCACGtagtcagcatgtgagctttgga forward oligo to amplify the OsPDS-TTV-crRNA02 target site

HTS-457-R TCGGCAatatcatacctgctccagcaat reverse oligo to amplify the OsPDS-TTV-crRNA02 target site

HTS-457-R1

TCGAAGatatcatacctgctccagcaat reverse oligo to amplify the OsPDS-TTV-crRNA02 target site

HTS-457-R2

TCCCGAatatcatacctgctccagcaat reverse oligo to amplify the OsPDS-TTV-crRNA02 target site

HTS-458-F TTAGGCtattttgttgacaggacaacgc forward oligo to amplify the OsROC5-TTV-crRNA01 target site

HTS-458-R TAATCGgttggcacagaggagaaggatg reverse oligo to amplify the OsROC5-TTV-crRNA01 target site

HTS-458-R1

GACGACgttggcacagaggagaaggatg reverse oligo to amplify the OsROC5-TTV-crRNA01 target site

HTS-458-R2

CTCAGAgttggcacagaggagaaggatg reverse oligo to amplify the OsROC5-TTV-crRNA01 target site

HTS-459-F TGACCAtagtaccaattagggaggttacattt forward oligo to amplify the OsROC5-TTV-crRNA02 target site

HTS-459-R CTATACgcaaacctgttcagaactacaaga reverse oligo to amplify the OsROC5-TTV-crRNA02 target site

HTS-459-R1

CTAGCTgcaaacctgttcagaactacaaga reverse oligo to amplify the OsROC5-TTV-crRNA02 target site

HTS-459-R2

CGGAATgcaaacctgttcagaactacaaga reverse oligo to amplify the OsROC5-TTV-crRNA02 target site

HTS-460-F ACAGTGtctgagttggccgttggcgtgc forward oligo to amplify the OsDEP1-TTV-crRNA01 target site

HTS-460-R CCAACAccgtactcggccgcttgggttt reverse oligo to amplify the OsDEP1-TTV-crRNA01 target site

HTS-460-R1

CATTTTccgtactcggccgcttgggttt reverse oligo to amplify the OsDEP1-TTV-crRNA01 target site

HTS-460-R2

CATGGCccgtactcggccgcttgggttt reverse oligo to amplify the OsDEP1-TTV-crRNA01 target site

HTS-461-F GCCAATgtgcatagtctattttacttcaacagg forward oligo to amplify the OsDEP1-TTV-

15

crRNA02 target site HTS-461-R CAGGCGcataagacattagataaggacagca reverse oligo to amplify the OsDEP1-TTV-

crRNA02 target site HTS-461-R1

CACTCAcataagacattagataaggacagca reverse oligo to amplify the OsDEP1-TTV-crRNA02 target site

HTS-461-R2

CACGATcataagacattagataaggacagca reverse oligo to amplify the OsDEP1-TTV-crRNA02 target site

HTS-462-F CAGATCggtacaaatatgccacccttca forward oligo to amplify the OsmiR528-TTV-crRNA01 target site

HTS-462-R CACCGGtgagagtttggtgcaataacagaaaagt reverse oligo to amplify the OsmiR528-TTV-crRNA01 target site

HTS-462-R1

CAACTAtgagagtttggtgcaataacagaaaagt reverse oligo to amplify the OsmiR528-TTV-crRNA01 target site

HTS-462-R2

CAAAAGtgagagtttggtgcaataacagaaaagt reverse oligo to amplify the OsmiR528-TTV-crRNA01 target site

HTS-463-F ACTTGAggtacaaatatgccacccttca forward oligo to amplify the OsmiR528-TTV-crRNA02 target site

HTS-463-R ATTCCTtgagagtttggtgcaataacagaaaagt reverse oligo to amplify the OsmiR528-TTTV-crRNA02 target site

HTS-463-R1

ATGAGCtgagagtttggtgcaataacagaaaagt reverse oligo to amplify the OsmiR528-TTTV-crRNA02 target site

HTS-463-R2

ACTGATtgagagtttggtgcaataacagaaaagt reverse oligo to amplify the OsmiR528-TTTV-crRNA02 target site

HTS-464-F GATCAGtatgcctggcatgagcagagcc forward oligo to amplify the OsEPFL9-TTTV-crRNA01 target site

HTS-464-R GGTAGCaggagaacagaagaaaagacaaga reverse oligo to amplify the OsEPFL9-TTTV-crRNA01 target site

HTS-464-R1

GAGTGGaggagaacagaagaaaagacaaga reverse oligo to amplify the OsEPFL9-TTTV-crRNA01 target site

HTS-464-R2

CGTACGaggagaacagaagaaaagacaaga reverse oligo to amplify the OsEPFL9-TTTV-crRNA01 target site

HTS-465-F TAGCTTgttgcttctctaatccaggcacac forward oligo to amplify the OsEPFL9-TTV-crRNA02 target site

HTS-465-R GTTTCGtgttcttcaggtaatgtttggtg reverse oligo to amplify the OsEPFL9-TTV-crRNA02 target site

HTS-465-R1

GTGGCCtgttcttcaggtaatgtttggtg reverse oligo to amplify the OsEPFL9-TTV-crRNA02 target site

HTS-465-R2

GTGAAAtgttcttcaggtaatgtttggtg reverse oligo to amplify the OsEPFL9-TTV-crRNA02 target site

HTS-466-F GGCTACgtacgacagtacttgctgtctagc forward oligo to amplify the OsGS3-TTV-crRNA01 target site

HTS-466-R GTCCGCaacggtcaaagttcatgatcaaaa reverse oligo to amplify the OsGS3-TTV-crRNA01 target site

HTS-466-R1

GTAGAGaacggtcaaagttcatgatcaaaa reverse oligo to amplify the OsGS3-TTV-crRNA01 target site

HTS-466-R2

CCGTCCaacggtcaaagttcatgatcaaaa reverse oligo to amplify the OsGS3-TTV-crRNA01 target site

HTS-467-F CTTGTAcctggtgaacttcgtcgattgtg forward oligo to amplify the OsGS3-TTV-crRNA02 target site

HTS-467-R ATGTCAatccgggaatgaagtgagtacc reverse oligo to amplify the OsGS3-TTV-crRNA02 target site

HTS-467-R1

AGTTCCatccgggaatgaagtgagtacc reverse oligo to amplify the OsGS3-TTV-crRNA02 target site

HTS-467-R2

AGTCAAatccgggaatgaagtgagtacc reverse oligo to amplify the OsGS3-TTV-crRNA02 target site

LR541-LR561-F

CACTCActggctgcctgtcatctatgaa forward oligo to amplify the LbCpf1-OsPDS-TCCG-crRNA01 target site

16

LR541-LR561-R1

CATGGCggaggtcttggaaagtcctgg reverse oligo to amplify the LbCpf1-OsPDS-TCCG-crRNA01 target site

LR541-LR561-R2

CATTTTggaggtcttggaaagtcctgg reverse oligo to amplify the LbCpf1-OsPDS-TCCG-crRNA01 target site

LR542-LR562-F

GGCTACggaaggatgaagatggagattg forward oligo to amplify the LbCpf1-OsPDS-TCCC-crRNA01 target site

LR542-LR562-R1

AGTCAAgctcatgatatttatgtgacgttaa reverse oligo to amplify the LbCpf1-OsPDS-TCCC-crRNA01 target site

LR542-LR562-R2

AGTTCCgctcatgatatttatgtgacgttaa reverse oligo to amplify the LbCpf1-OsPDS-TCCC-crRNA01 target site

LR543-LR563-F

CACTCActggctgcctgtcatctatgaa forward oligo to amplify the LbCpf1-OsPDS-TTCC-crRNA01 target site

LR543-LR563-R1

CATGGCggaggtcttggaaagtcctgg reverse oligo to amplify the LbCpf1-OsPDS-TTCC-crRNA01 target site

LR543-LR563-R2

CATTTTggaggtcttggaaagtcctgg reverse oligo to amplify the LbCpf1-OsPDS-TTCC-crRNA01 target site

LR544-LR564-F

GGCTACggaaggatgaagatggagattg forward oligo to amplify the LbCpf1-OsPDS-TTCA-crRNA01 target site

LR544-LR564-R1

AGTCAAgctcatgatatttatgtgacgttaa reverse oligo to amplify the LbCpf1-OsPDS-TTCA-crRNA01 target site

LR544-LR564-R2

AGTTCCgctcatgatatttatgtgacgttaa reverse oligo to amplify the LbCpf1-OsPDS-TTCA-crRNA01 target site

LR545-LR565-F

GGCTACggaaggatgaagatggagattg forward oligo to amplify the LbCpf1-OsPDS-TATC-crRNA01 target site

LR545-LR565-R1

AGTCAAgctcatgatatttatgtgacgttaa reverse oligo to amplify the LbCpf1-OsPDS-TATC-crRNA01 target site

LR545-LR565-R2

AGTTCCgctcatgatatttatgtgacgttaa reverse oligo to amplify the LbCpf1-OsPDS-TATC-crRNA01 target site

LR546-LR566-F

GGCTACggaaggatgaagatggagattg forward oligo to amplify the LbCpf1-OsPDS-TATG-crRNA01 target site

LR546-LR566-R1

AGTCAAgctcatgatatttatgtgacgttaa reverse oligo to amplify the LbCpf1-OsPDS-TATG-crRNA01 target site

LR546-LR566-R2

AGTTCCgctcatgatatttatgtgacgttaa reverse oligo to amplify the LbCpf1-OsPDS-TATG-crRNA01 target site

LR547-LR567-HTS-F

CCAACATTCAGCCGGTTTGATTTTCCTG forward oligo to amplify the LbCpf1-OsPDS-CCCC-crRNA01 target site

LR547-LR567-HTS-R

CGGAATGGCCAAGTTAGCATTTCATTGTTTCT reverse oligo to amplify the LbCpf1-OsPDS-CCCC-crRNA01 target site

LR547-LR567-HTS-R1

CTAGCTGGCCAAGTTAGCATTTCATTGTTTCT reverse oligo to amplify the LbCpf1-OsPDS-CCCC-crRNA01 target site

LR547-LR567-HTS-R2

CTATACGGCCAAGTTAGCATTTCATTGTTTCT reverse oligo to amplify the LbCpf1-OsPDS-CCCC-crRNA01 target site

LR548-LR568-F

GGTAGCgctgctggtgagtgctgat forward oligo to amplify the LbCpf1-OsROC5-TCCG-crRNA01 target site

LR548-LR568-R1

ATGAGCacccattgggagtgtcttgc reverse oligo to amplify the LbCpf1-OsROC5-TCCG-crRNA01 target site

LR548-LR568-R2

ATTCCTacccattgggagtgtcttgc reverse oligo to amplify the LbCpf1-OsROC5-TCCG-crRNA01 target site

LR549-LR569-F

CTCAGAgtgtaagacgtagctaacatattttctt forward oligo to amplify the LbCpf1-OsROC5-TCCA-crRNA01 target site

LR549-LR569-R1

ACTGATtgacaatactattaactgacttactgcc reverse oligo to amplify the LbCpf1-OsROC5-TCCA-crRNA01 target site

LR549- ATGAGCtgacaatactattaactgacttactgcc reverse oligo to amplify the LbCpf1-OsROC5-

17

LR569-R2 TCCA-crRNA01 target site LR550-LR570-F

GTGGCCaggtttgggctaatgtcctcc forward oligo to amplify the LbCpf1-OsROC5-TTCC-crRNA01 target site

LR550-LR570-R1

CGTACGcacctgtagctcagccttcat reverse oligo to amplify the LbCpf1-OsROC5-TTCC-crRNA01 target site

LR550-LR570-R2

GAGTGGcacctgtagctcagccttcat reverse oligo to amplify the LbCpf1-OsROC5-TTCC-crRNA01 target site

LR552-LR572-F

CTCAGAgtgtaagacgtagctaacatattttctt forward oligo to amplify the LbCpf1-OsROC5-TATG-crRNA01 target site

LR552-LR572-R1

ACTGATtgacaatactattaactgacttactgcc reverse oligo to amplify the LbCpf1-OsROC5-TATG-crRNA01 target site

LR552-LR572-R2

ATGAGCtgacaatactattaactgacttactgcc reverse oligo to amplify the LbCpf1-OsROC5-TATG-crRNA01 target site

LR553-LR573-F

ATTCCTtcgtgtgccttagtcttcttctc forward oligo to amplify the LbCpf1-OsROC5-CCCC-crRNA01 target site

LR553-LR573-R1

CGATGTTAGGAACTTGGTGGCGAGG reverse oligo to amplify the LbCpf1-OsROC5-CCCC-crRNA01 target site

LR553-LR573-R2

TTAGGCTAGGAACTTGGTGGCGAGG reverse oligo to amplify the LbCpf1-OsROC5-CCCC-crRNA01 target site

LR554-LR574-F

CAAAAGtggtacaaaacatgacccact forward oligo to amplify the LbCpf1-OsDEP1-TCCA-crRNA01 target site

LR554-LR574-R1

CACCGGgggactagaggcactttcaga reverse oligo to amplify the LbCpf1-OsDEP1-TCCA-crRNA01 target site

LR554-LR574-R2

CACGATgggactagaggcactttcaga reverse oligo to amplify the LbCpf1-OsDEP1-TCCA-crRNA01 target site

LR555-LR575-F

TCGGCAgcggctgaaattacatgtagaaac forward oligo to amplify the LbCpf1-OsDEP1-TCCC-crRNA01 target site

LR555-LR575-R1

TCCCGAttggacaggagcacgagcagtt reverse oligo to amplify the LbCpf1-OsDEP1-TCCC-crRNA01 target site

LR555-LR575-R2

TCATTCttggacaggagcacgagcagtt reverse oligo to amplify the LbCpf1-OsDEP1-TCCC-crRNA01 target site

LR556-LR576-F

CAAAAGtggtacaaaacatgacccact forward oligo to amplify the LbCpf1-OsDEP1-TTCC-crRNA01 target site

LR556-LR576-R1

CACCGGgggactagaggcactttcaga reverse oligo to amplify the LbCpf1-OsDEP1-TTCC-crRNA01 target site

LR556-LR576-R2

CACGATgggactagaggcactttcaga reverse oligo to amplify the LbCpf1-OsDEP1-TTCC-crRNA01 target site

LR557-LR577-F

TCGGCAgcggctgaaattacatgtagaaac forward oligo to amplify the LbCpf1-OsDEP1-TTCA-crRNA01 target site

LR557-LR577-R1

TATAATttggacaggagcacgagcagtt reverse oligo to amplify the LbCpf1-OsDEP1-TTCA-crRNA01 target site

LR557-LR577-R2

TACAGCttggacaggagcacgagcagtt reverse oligo to amplify the LbCpf1-OsDEP1-TTCA-crRNA01 target site

LR558-LR578-F

CAAAAGtggtacaaaacatgacccact forward oligo to amplify the LbCpf1-OsDEP1-TATG-crRNA01 target site

LR558-LR578-R1

CACCGGgggactagaggcactttcaga reverse oligo to amplify the LbCpf1-OsDEP1-TATG-crRNA01 target site

LR558-LR578-R2

CACGATgggactagaggcactttcaga reverse oligo to amplify the LbCpf1-OsDEP1-TATG-crRNA01 target site

LR559-LR579-F

TCGGCAgcggctgaaattacatgtagaaac forward oligo to amplify the LbCpf1-OsDEP1-TATC-crRNA01 target site

LR559-LR579-R1

TAATCGttggacaggagcacgagcagtt reverse oligo to amplify the LbCpf1-OsDEP1-TATC-crRNA01 target site

LR559-LR579-R2

GACGACttggacaggagcacgagcagtt reverse oligo to amplify the LbCpf1-OsDEP1-TATC-crRNA01 target site

LR560-LR580-F

CAAAAGtggtacaaaacatgacccact forward oligo to amplify the LbCpf1-OsDEP1-CCCC-crRNA01 target site

18

LR560-LR580-R1

CACCGGgggactagaggcactttcaga reverse oligo to amplify the LbCpf1-OsDEP1-CCCC-crRNA01 target site

LR560-LR580-R2

CACGATgggactagaggcactttcaga reverse oligo to amplify the LbCpf1-OsDEP1-CCCC-crRNA01 target site

LR541-LR561-F

CACTCActggctgcctgtcatctatgaa forward oligo to amplify the FnCpf1-OsPDS-TCCG-crRNA01 target site

LR541-LR561-R1

CATGGCggaggtcttggaaagtcctgg reverse oligo to amplify the FnCpf1-OsPDS-TCCG-crRNA01 target site

LR541-LR561-R2

CATTTTggaggtcttggaaagtcctgg reverse oligo to amplify the FnCpf1-OsPDS-TCCG-crRNA01 target site

LR542-LR562-F

GGCTACggaaggatgaagatggagattg forward oligo to amplify the FnCpf1-OsPDS-TCCC-crRNA01 target site

LR542-LR562-R1

AGTCAAgctcatgatatttatgtgacgttaa reverse oligo to amplify the FnCpf1-OsPDS-TCCC-crRNA01 target site

LR542-LR562-R2

AGTTCCgctcatgatatttatgtgacgttaa reverse oligo to amplify the FnCpf1-OsPDS-TCCC-crRNA01 target site

LR543-LR563-F

CACTCActggctgcctgtcatctatgaa forward oligo to amplify the FnCpf1-OsPDS-TTCC-crRNA01 target site

LR543-LR563-R1

CATGGCggaggtcttggaaagtcctgg reverse oligo to amplify the FnCpf1-OsPDS-TTCC-crRNA01 target site

LR543-LR563-R2

CATTTTggaggtcttggaaagtcctgg reverse oligo to amplify the FnCpf1-OsPDS-TTCC-crRNA01 target site

LR544-LR564-F

GGCTACggaaggatgaagatggagattg forward oligo to amplify the FnCpf1-OsPDS-TTCA-crRNA01 target site

LR544-LR564-R1

AGTCAAgctcatgatatttatgtgacgttaa reverse oligo to amplify the FnCpf1-OsPDS-TTCA-crRNA01 target site

LR544-LR564-R2

AGTTCCgctcatgatatttatgtgacgttaa reverse oligo to amplify the FnCpf1-OsPDS-TTCA-crRNA01 target site

LR545-LR565-F

GGCTACggaaggatgaagatggagattg forward oligo to amplify the FnCpf1-OsPDS-TATC-crRNA01 target site

LR545-LR565-R1

AGTCAAgctcatgatatttatgtgacgttaa reverse oligo to amplify the FnCpf1-OsPDS-TATC-crRNA01 target site

LR545-LR565-R2

AGTTCCgctcatgatatttatgtgacgttaa reverse oligo to amplify the FnCpf1-OsPDS-TATC-crRNA01 target site

LR546-LR566-F

GGCTACggaaggatgaagatggagattg forward oligo to amplify the FnCpf1-OsPDS-TATG-crRNA01 target site

LR546-LR566-R1

AGTCAAgctcatgatatttatgtgacgttaa reverse oligo to amplify the FnCpf1-OsPDS-TATG-crRNA01 target site

LR546-LR566-R2

AGTTCCgctcatgatatttatgtgacgttaa reverse oligo to amplify the FnCpf1-OsPDS-TATG-crRNA01 target site

LR547-LR567-HTS-F

CCAACATTCAGCCGGTTTGATTTTCCTG forward oligo to amplify the FnCpf1-OsPDS-CCCC-crRNA01 target site

LR547-LR567-HTS-R

CGGAATGGCCAAGTTAGCATTTCATTGTTTCT reverse oligo to amplify the FnCpf1-OsPDS-CCCC-crRNA01 target site

LR547-LR567-HTS-R1

CTAGCTGGCCAAGTTAGCATTTCATTGTTTCT reverse oligo to amplify the FnCpf1-OsPDS-CCCC-crRNA01 target site

LR547-LR567-HTS-R2

CTATACGGCCAAGTTAGCATTTCATTGTTTCT reverse oligo to amplify the FnCpf1-OsPDS-CCCC-crRNA01 target site

LR548-LR568-F

GGTAGCgctgctggtgagtgctgat forward oligo to amplify the FnCpf1-OsROC5-TCCG-crRNA01 target site

LR548-LR568-R1

ATGAGCacccattgggagtgtcttgc reverse oligo to amplify the FnCpf1-OsROC5-TCCG-crRNA01 target site

LR548- ATTCCTacccattgggagtgtcttgc reverse oligo to amplify the FnCpf1-OsROC5-

19

LR568-R2 TCCG-crRNA01 target site LR549-LR569-F

CTCAGAgtgtaagacgtagctaacatattttctt forward oligo to amplify the FnCpf1-OsROC5-TCCA-crRNA01 target site

LR549-LR569-R1

ACTGATtgacaatactattaactgacttactgcc reverse oligo to amplify the FnCpf1-OsROC5-TCCA-crRNA01 target site

LR549-LR569-R2

ATGAGCtgacaatactattaactgacttactgcc reverse oligo to amplify the FnCpf1-OsROC5-TCCA-crRNA01 target site

LR550-LR570-F

GTGGCCaggtttgggctaatgtcctcc forward oligo to amplify the FnCpf1-OsROC5-TTCC-crRNA01 target site

LR550-LR570-R1

CGTACGcacctgtagctcagccttcat reverse oligo to amplify the FnCpf1-OsROC5-TTCC-crRNA01 target site

LR550-LR570-R2

GAGTGGcacctgtagctcagccttcat reverse oligo to amplify the FnCpf1-OsROC5-TTCC-crRNA01 target site

LR552-LR572-F

CTCAGAgtgtaagacgtagctaacatattttctt forward oligo to amplify the FnCpf1-OsROC5-TATG-crRNA01 target site

LR552-LR572-R1

ACTGATtgacaatactattaactgacttactgcc reverse oligo to amplify the FnCpf1-OsROC5-TATG-crRNA01 target site

LR552-LR572-R2

ATGAGCtgacaatactattaactgacttactgcc reverse oligo to amplify the FnCpf1-OsROC5-TATG-crRNA01 target site

LR553-LR573-F

ATTCCTtcgtgtgccttagtcttcttctc forward oligo to amplify the FnCpf1-OsROC5-CCCC-crRNA01 target site

LR553-LR573-R1

CGATGTTAGGAACTTGGTGGCGAGG reverse oligo to amplify the FnCpf1-OsROC5-CCCC-crRNA01 target site

LR553-LR573-R2

TTAGGCTAGGAACTTGGTGGCGAGG reverse oligo to amplify the FnCpf1-OsROC5-CCCC-crRNA01 target site

LR554-LR574-F

CAAAAGtggtacaaaacatgacccact forward oligo to amplify the FnCpf1-OsDEP1-TCCA-crRNA01 target site

LR554-LR574-R1

CACCGGgggactagaggcactttcaga reverse oligo to amplify the FnCpf1-OsDEP1-TCCA-crRNA01 target site

LR554-LR574-R2

CACGATgggactagaggcactttcaga reverse oligo to amplify the FnCpf1-OsDEP1-TCCA-crRNA01 target site

LR555-LR575-F

TCGGCAgcggctgaaattacatgtagaaac forward oligo to amplify the FnCpf1-OsDEP1-TCCC-crRNA01 target site

LR555-LR575-R1

TCCCGAttggacaggagcacgagcagtt reverse oligo to amplify the FnCpf1-OsDEP1-TCCC-crRNA01 target site

LR555-LR575-R2

TCATTCttggacaggagcacgagcagtt reverse oligo to amplify the FnCpf1-OsDEP1-TCCC-crRNA01 target site

LR556-LR576-F

CAAAAGtggtacaaaacatgacccact forward oligo to amplify the FnCpf1-OsDEP1-TTCC-crRNA01 target site

LR556-LR576-R1

CACCGGgggactagaggcactttcaga reverse oligo to amplify the FnCpf1-OsDEP1-TTCC-crRNA01 target site

LR556-LR576-R2

CACGATgggactagaggcactttcaga reverse oligo to amplify the FnCpf1-OsDEP1-TTCC-crRNA01 target site

LR557-LR577-F

TCGGCAgcggctgaaattacatgtagaaac forward oligo to amplify the FnCpf1-OsDEP1-TTCA-crRNA01 target site

LR557-LR577-R1

TATAATttggacaggagcacgagcagtt reverse oligo to amplify the FnCpf1-OsDEP1-TTCA-crRNA01 target site

LR557-LR577-R2

TACAGCttggacaggagcacgagcagtt reverse oligo to amplify the FnCpf1-OsDEP1-TTCA-crRNA01 target site

LR558-LR578-F

CAAAAGtggtacaaaacatgacccact forward oligo to amplify the FnCpf1-OsDEP1-TATG-crRNA01 target site

LR558-LR578-R1

CACCGGgggactagaggcactttcaga reverse oligo to amplify the FnCpf1-OsDEP1-TATG-crRNA01 target site

LR558-LR578-R2

CACGATgggactagaggcactttcaga reverse oligo to amplify the FnCpf1-OsDEP1-TATG-crRNA01 target site

LR559-LR579-F

TCGGCAgcggctgaaattacatgtagaaac forward oligo to amplify the FnCpf1-OsDEP1-TATC-crRNA01 target site

20

LR559-LR579-R1

TAATCGttggacaggagcacgagcagtt reverse oligo to amplify the FnCpf1-OsDEP1-TATC-crRNA01 target site

LR559-LR579-R2

GACGACttggacaggagcacgagcagtt reverse oligo to amplify the FnCpf1-OsDEP1-TATC-crRNA01 target site

LR560-LR580-F

CAAAAGtggtacaaaacatgacccact forward oligo to amplify the FnCpf1-OsDEP1-CCCC-crRNA01 target site

LR560-LR580-R1

CACCGGgggactagaggcactttcaga reverse oligo to amplify the FnCpf1-OsDEP1-CCCC-crRNA01 target site

LR560-LR580-R2

CACGATgggactagaggcactttcaga reverse oligo to amplify the FnCpf1-OsDEP1-CCCC-crRNA01 target site

Oligos for vector construction230-RR-F1 ACCCCCAGTTCATGCGCGGCTGGGACAAGGACAAGGAAACGGA Constructing entry clone for Cpf1 with

altered PAM230-RR-R1 TTGCTGAAGAAAACCCTTGGGAGCATCTTGTTCGGACCCGGAAGC Constructing entry clone for Cpf1 with

altered PAM230-RR-F2 ACAAGATGCTCCCAAGGGTTTTCTTCAGCAAGAAATGGATGGCCT Constructing entry clone for Cpf1 with

altered PAM230-RR-R2 CCTTGTCCCAGCCGCGCATGAACTGGGGGTTCTGGAAGTAGAG Constructing entry clone for Cpf1 with

altered PAM239-RR-F1 ATAGTACTCTTGCCCGCGGCTGGGACAAGAATAAGGAGCCAGAT

AConstructing entry clone for Cpf1 with altered PAM

239-RR-R1 GCCGAAAAAAAAACCCTGGGGAGCATTTTATTTGCTCCCGGCAAC Constructing entry clone for Cpf1 with altered PAM

239-RR-F2 ATAAAATGCTCCCCAGGGTTTTTTTTTCGGCAAAGAGCATTAAAT Constructing entry clone for Cpf1 with altered PAM

239-RR-R2 TTCTTGTCCCAGCCGCGGGCAAGAGTACTATTTTCGAAATTGAGC Constructing entry clone for Cpf1 with altered PAM

SpnR-F1 GACATTGATCTGGCTATCTTGCT Constructing entry clone for Cpf1 with altered PAM

230-RVR-R1

CGATCCGTTTCCACGTCCTTGTCCCAGCCGCGCATGAACTGGGGGTTCTGGAAGTAGAG

Constructing entry clone for Cpf1 with altered PAM

230-RVR-F2

CGGCTGGGACAAGGACGTGGAAACGGATCGCAGGGCTACGATCCTGAGGTATGGTTCA


SpnR-R2 CGCTGGACCTACCAAGGCAACG Constructing entry clone for Cpf1 with altered PAM

239-RVR-R1

CCTATCTGGCTCCACATTCTTGTCCCAGCCGCGGGCAAGAGTACTATTTTCGAAATTGA


239-RVR-F2

CGCGGCTGGGACAAGAATGTGGAGCCAGATAGGACCGCCATACTTTTTATCAAAGATGA


M13-F1 TTCCCAGTCACGACGTTGTAAAAC Sequencing Cpf1 expression entry cloneM13-R1 TTTGAGACACGGGCCAGAGCTGC Sequencing Cpf1 expression entry cloneCpf1-seq1 GGCACCGTCACGACCACAGAGC Sequencing Cpf1 expression entry cloneCpf1-seq2 TCCGCAACGAGAATGTGCTC Sequencing Cpf1 expression entry cloneCpf1-seq3 GGGTGGGACGTTAACAAAGAGAAG Sequencing Cpf1 expression entry cloneCpf1-seq4 GAACTCAACCCACTCCTGTATCAC Sequencing Cpf1 expression entry cloneCpf1-seq5 AGCGGAACCTCATCTACATCAC Sequencing Cpf1 expression entry cloneCpf1-seq6 GGTTTCGACTTCCTGCACTATGAT Sequencing Cpf1 expression entry clone239-seq1 GCGATGACGACAATCTCCAG Sequencing Cpf1 expression entry clone239-seq2 TTCACTCGACGAGGTGTTTG Sequencing Cpf1 expression entry clone239-seq3 CCCTCTAAGAAAGAACAGGAGTTG Sequencing Cpf1 expression entry clone239-seq4 CCAACGGCTGGGACAAGAATAAG Sequencing Cpf1 expression entry clone239-seq5 TCAGTGGTCAATCAGGGCAAAC Sequencing Cpf1 expression entry clone239-seq6 TGGCATACTACACCCTTGTTG Sequencing Cpf1 expression entry clone239-seq7 CCAAATACGAATCCGTGAGCAAG Sequencing Cpf1 expression entry cloneFn-crRNA- GATCCAAATTACTGATGAGTCCGTGAGGACGAAACGAGTAAGCTC Constructing crRNA cloning vector for FnCpf1

21

F GTCTAATTTCTACTGTTGTAGATGAGACGGAGCTFn-crRNA-R

CCGTCTCATCTACAACAGTAGAAATTAGACGAGCTTACTCGTTTCGTCCTCACGGACTCATCAGTAATTTG

Constructing crRNA cloning vector for FnCpf1

Ubi-intron-F1

CCCTGTTGTTTGGTGTTACTTC Sequencing crRNA expression entry clone

Cpf1-OsPDS-gR1-F

TAGATGAGTGAAATCTCTTGTCTTAAGG Constructing crRNA expression entry clone

Cpf1-OsPDS-gR1-R

GGCCCCTTAAGACAAGAGATTTCACTCA Constructing crRNA expression entry clone

Cpf1-OsPDS-gR2-F

TAGATTTCAAAACCCTTAGAGATATCTA Constructing crRNA expression entry clone

Cpf1-OsPDS-gR2-R

GGCCTAGATATCTCTAAGGGTTTTGAAA Constructing crRNA expression entry clone

Cpf1-OsDEP1-gR1-F

TAGATCTACTGTTGCAAGTGCTCACCCA Constructing crRNA expression entry clone

Cpf1-OsDEP1-gR1-R

GGCCTGGGTGAGCACTTGCAACAGTAGA Constructing crRNA expression entry clone

Cpf1-OsDEP1-gR2-F

TAGATCAGAAAGAGAAGGAGGCACAGAT Constructing crRNA expression entry clone

Cpf1-OsDEP1-gR2-R

GGCCATCTGTGCCTCCTTCTCTTTCTGA Constructing crRNA expression entry clone

Cpf1-OsROC5-gR1-F

TAGATTGCTTCCTGCAATGCCGGTAGAC Constructing crRNA expression entry clone

Cpf1-OsROC5-gR1-R

GGCCGTCTACCGGCATTGCAGGAAGCAA Constructing crRNA expression entry clone

Cpf1-OsROC5-gR2-F

TAGATTAAGCAGCTGGCTGAGGGTGCAT Constructing crRNA expression entry clone

Cpf1-OsROC5-gR2-R

GGCCATGCACCCTCAGCCAGCTGCTTAA Constructing crRNA expression entry clone

TTN-OsPDS-crRNA1-F

TAGATAGGAATAAAGGAAAAAGATTCCG Constructing crRNA expression entry clone

TTN-OsPDS-crRNA1-R

GGCCCGGAATCTTTTTCCTTTATTCCTA Constructing crRNA expression entry clone

TTN-OsPDS-crRNA2-F

TAGATGAAAGTCCTGGCAAACAACCTGC Constructing crRNA expression entry clone

TTN-OsPDS-crRNA2-R

GGCCGCAGGTTGTTTGCCAGGACTTTCA Constructing crRNA expression entry clone

TTN-OsROC5-crRNA1-F

TAGATCTGGGCTCGGCGTCCTCGACGTC Constructing crRNA expression entry clone

TTN- GGCCGACGTCGAGGACGCCGAGCCCAGA Constructing crRNA expression entry clone

22

OsROC5-crRNA1-RTTN-OsROC5-crRNA2-F

TAGATCATCACACAGCCGGAAGGTACCC Constructing crRNA expression entry clone

TTN-OsROC5-crRNA2-R

GGCCGGGTACCTTCCGGCTGTGTGATGA Constructing crRNA expression entry clone

TTN-OsDEP1-crRNA1-F

TAGATCTCAAGGTGAGCGCCCCGCGGCG Constructing crRNA expression entry clone

TTN-OsDEP1-crRNA1-R

GGCCCGCCGCGGGGCGCTCACCTTGAGA Constructing crRNA expression entry clone

TTN-OsDEP1-crRNA2-F

TAGATACTTCCTTGAAGGAGCTCAGCCC Constructing crRNA expression entry clone

TTN-OsDEP1-crRNA2-R

GGCCGGGCTGAGCTCCTTCAAGGAAGTA Constructing crRNA expression entry clone

TTN-OsmiR528-crRNA1-F

TAGATAAACTGAATCTCCTGCTCCTCTG Constructing crRNA expression entry clone

TTN-OsmiR528-crRNA1-R

GGCCCAGAGGAGCAGGAGATTCAGTTTA Constructing crRNA expression entry clone

TTTN-OsmiR528-crRNA2-F

TAGATCCTCTCTCTCCTGTGCTTGCCTC Constructing crRNA expression entry clone

TTTN-OsmiR528-crRNA2-R

GGCCGAGGCAAGCACAGGAGAGAGAGGA Constructing crRNA expression entry clone

TTTN-OsEPFL9-crRNA1-F

TAGATAAGAAGGGTTATGGCCAATGCTT Constructing crRNA expression entry clone

TTTN-OsEPFL9-crRNA1-R

GGCCAAGCATTGGCCATAACCCTTCTTA Constructing crRNA expression entry clone

TTN-OsEPFL9-crRNA2-F

TAGATCAATCAAGGGCACCATGGCAGCA Constructing crRNA expression entry clone

TTN-OsEPFL9-crRNA2-R

GGCCTGCTGCCATGGTGCCCTTGATTGA Constructing crRNA expression entry clone

TTN-OsGS3-crRNA1-F

TAGATGATTGAATTTATTTCACCCTGCA Constructing crRNA expression entry clone

TTN-OsGS3-crRNA1-R

GGCCTGCAGGGTGAAATAAATTCAATCA Constructing crRNA expression entry clone

TTN-OsGS3-crRNA2-F

TAGATATCGGAAGAACTCCTGATCCATT Constructing crRNA expression entry clone

TTN-OsGS3-crRNA2-R

GGCCAATGGATCAGGAGTTCTTCCGATA Constructing crRNA expression entry clone

TCCG- TAGATACGGAATCTTTTTCCTTTATTCC Constructing crRNA expression entry clone

23

PDS-gR1-FTCCG-PDS-gR1-R

GGCCGGAATAAAGGAAAAAGATTCCGTA Constructing crRNA expression entry clone

TCCC-PDS-gR2-F

TAGATGCATGGCATTTCTACCTTATCGA Constructing crRNA expression entry clone

TCCC-PDS-gR2-R

GGCCTCGATAAGGTAGAAATGCCATGCA Constructing crRNA expression entry clone

TTCC-PDS-gR3-F

TAGATTTTATTCCTTAAGACAAGAGATT Constructing crRNA expression entry clone

TTCC-PDS-gR3-R

GGCCAATCTCTTGTCTTAAGGAATAAAA Constructing crRNA expression entry clone

TTCA-PDS-gR4-F

TAGATTACTTCCCGCATGGCATTTCTAC Constructing crRNA expression entry clone

TTCA-PDS-gR4-R

GGCCGTAGAAATGCCATGCGGGAAGTAA Constructing crRNA expression entry clone

TATC-PDS-gR5-F

TAGATAAACTCTTTCATCGATAAGGTAG Constructing crRNA expression entry clone

TATC-PDS-gR5-R

GGCCCTACCTTATCGATGAAAGAGTTTA Constructing crRNA expression entry clone

TATG-PDS-gR6-F

TAGATAAGTGATGTCTGAGGATTAACAC Constructing crRNA expression entry clone

TATG-PDS-gR6-R

GGCCGTGTTAATCCTCAGACATCACTTA Constructing crRNA expression entry clone

CCCC-PDS-gR7-F

TAGATTAATTACAGTCATGTGTTAGTAA Constructing crRNA expression entry clone

CCCC-PDS-gR7-R

GGCCTTACTAACACATGACTGTAATTAA Constructing crRNA expression entry clone

TCCG-ROC-gR1-F

TAGATGTTTTGTAAGCAGCTGGCTGAGG Constructing crRNA expression entry clone

TCCG-ROC-gR1-R

GGCCCCTCAGCCAGCTGCTTACAAAACA Constructing crRNA expression entry clone

TCCA-ROC-gR2-F

TAGATTTGATGGTTTAGTGAGAGATCAT Constructing crRNA expression entry clone

TCCA-ROC-gR2-R

GGCCATGATCTCTCACTAAACCATCAAA Constructing crRNA expression entry clone

TTCC-ROC-gR3-F

TAGATTGCAATGCCGGTAGACACCTCCT Constructing crRNA expression entry clone

TTCC-ROC-gR3-R

GGCCAGGAGGTGTCTACCGGCATTGCAA Constructing crRNA expression entry clone

TTCA-ROC-gR4-F

TAGATTCAGCAGCAACGCGCCATTTCTG Constructing crRNA expression entry clone

TTCA-ROC-gR4-R

GGCCCAGAAATGGCGCGTTGCTGCTGAA Constructing crRNA expression entry clone

TATG-ROC-gR5-F

TAGATATCTCTCACTAAACCATCAATGG Constructing crRNA expression entry clone

TATG-ROC-gR5-R

GGCCCCATTGATGGTTTAGTGAGAGATA Constructing crRNA expression entry clone

24

CCCC-ROC-gR6-F

TAGATGCCATGCTCGGGGAGGTGTCCCT Constructing crRNA expression entry clone

CCCC-ROC-gR6-R

GGCCAGGGACACCTCCCCGAGCATGGCA Constructing crRNA expression entry clone

TCCA-DEP-gR1-F

TAGATGAAAGAGAAGGAGGCACAGATCT Constructing crRNA expression entry clone

TCCA-DEP-gR1-R

GGCCAGATCTGTGCCTCCTTCTCTTTCA Constructing crRNA expression entry clone

TCCC-DEP-gR2-F

TAGATTGCAGATCAAAATTGTGTATCTG Constructing crRNA expression entry clone

TCCC-DEP-gR2-R

GGCCCAGATACACAATTTTGATCTGCAA Constructing crRNA expression entry clone

TTCC-DEP-gR3-F

TAGATAGAAAGAGAAGGAGGCACAGATC Constructing crRNA expression entry clone

TTCC-DEP-gR3-R

GGCCGATCTGTGCCTCCTTCTCTTTCTA Constructing crRNA expression entry clone

TTCA-DEP-gR4-F

TAGATTGTCTTTGCTACTGTTGCAAGTG Constructing crRNA expression entry clone

TTCA-DEP-gR4-R

GGCCCACTTGCAACAGTAGCAAAGACAA Constructing crRNA expression entry clone

TATG-DEP-gR5-F

TAGATTTTTGATCCAATATAGTTTGCTC Constructing crRNA expression entry clone

TATG-DEP-gR5-R

GGCCGAGCAAACTATATTGGATCAAAAA Constructing crRNA expression entry clone

TATC-DEP-gR6-F

TAGATTGCATTTCATGTCTTTGCTACTG Constructing crRNA expression entry clone

TATC-DEP-gR6-R

GGCCCAGTAGCAAAGACATGAAATGCAA Constructing crRNA expression entry clone

CCCC-DEP-gR7-F

TAGATTCAGAACCTGCGAGCAAACTATA Constructing crRNA expression entry clone

CCCC-DEP-gR7-R

GGCCTATAGTTTGCTCGCAGGTTCTGAA Constructing crRNA expression entry clone

TTN-OsROC5-crRNA1-MS1-F

TAGATGAGGGCTCGGCGTCCTCGACGTC Constructing crRNA expression entry clone

TTN-OsROC5-crRNA1-MS1-R

GGCCGACGTCGAGGACGCCGAGCCCTCA Constructing crRNA expression entry clone


TAGATCTGGATTCGGCGTCCTCGACGTC Constructing crRNA expression entry clone


GGCCGACGTCGAGGACGCCGAATCCAGA Constructing crRNA expression entry clone


TAGATCTGGGCTCCCCGTCCTCGACGTC Constructing crRNA expression entry clone

TTN-OsROC5-crRNA1-

GGCCGACGTCGAGGACGGGGAGCCCAGA Constructing crRNA expression entry clone

25

MS3-RTTN-OsROC5-crRNA1-MS4-F

TAGATCTGGGCTCGGCGAGCTCGACGTC Constructing crRNA expression entry clone


GGCCGACGTCGAGCTCGCCGAGCCCAGA Constructing crRNA expression entry clone


TAGATCTGGGCTCGGCGTCCTATACGTC Constructing crRNA expression entry clone


GGCCGACGTATAGGACGCCGAGCCCAGA Constructing crRNA expression entry clone


TAGATCTGGGCTCGGCGTCCTCGACCAC Constructing crRNA expression entry clone


GGCCGTGGTCGAGGACGCCGAGCCCAGA Constructing crRNA expression entry clone

TTTN-OsEPFL9-crRNA1-MS1-F

TAGATTTGAAGGGTTATGGCCAATGCTT Constructing crRNA expression entry clone

TTTN-OsEPFL9-crRNA1-MS1-R

GGCCAAGCATTGGCCATAACCCTTCAAA Constructing crRNA expression entry clone


TAGATAAGATCGGTTATGGCCAATGCTT Constructing crRNA expression entry clone


GGCCAAGCATTGGCCATAACCGATCTTA Constructing crRNA expression entry clone


TAGATAAGAAGGGAAATGGCCAATGCTT Constructing crRNA expression entry clone


GGCCAAGCATTGGCCATTTCCCTTCTTA Constructing crRNA expression entry clone


TAGATAAGAAGGGTTATTACCAATGCTT Constructing crRNA expression entry clone


GGCCAAGCATTGGTAATAACCCTTCTTA Constructing crRNA expression entry clone

TTTN- TAGATAAGAAGGGTTATGGCCTCTGCTT Constructing crRNA expression entry clone

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OsEPFL9-crRNA1-MS5-FTTTN-OsEPFL9-crRNA1-MS5-R

GGCCAAGCAGAGGCCATAACCCTTCTTA Constructing crRNA expression entry clone


TAGATAAGAAGGGTTATGGCCAATGGAT Constructing crRNA expression entry clone


GGCCATCCATTGGCCATAACCCTTCTTA Constructing crRNA expression entry clone

AA-crRNA1-F

TAGATTCAGGTTCAGAATGCTGGTCCCC Constructing crRNA expression entry clone

AA-crRNA1-R

GGCCGGGGACCAGCATTCTGAACCTGAA Constructing crRNA expression entry clone

AA-crRNA2-F

TAGATCCGGTGAAAAGGACCTTGTCCCA Constructing crRNA expression entry clone

AA-crRNA2-R

GGCCTGGGACAAGGTCCTTTTCACCGGA Constructing crRNA expression entry clone

AC-crRNA1-F

TAGATTGGGGCCTTGCAAGGTCACCTCC Constructing crRNA expression entry clone

AC-crRNA1-R

GGCCGGAGGTGACCTTGCAAGGCCCCAA Constructing crRNA expression entry clone

AC-crRNA2-F

TAGATGTCTCCCTGCAAACCACCGCGCA Constructing crRNA expression entry clone

AC-crRNA2-R

GGCCTGCGCGGTGGTTTGCAGGGAGACA Constructing crRNA expression entry clone

AG-crRNA1-F

TAGATCCGGCAGCTAATAGGGATCTAAA Constructing crRNA expression entry clone

AG-crRNA1-R

GGCCTTTAGATCCCTATTAGCTGCCGGA Constructing crRNA expression entry clone

AG-crRNA2-F

TAGATGGGCATGGAGACAGGAGACATAG Constructing crRNA expression entry clone

AG-crRNA2-R

GGCCCTATGTCTCCTGTCTCCATGCCCA Constructing crRNA expression entry clone

CA-crRNA1-F

TAGATTACGTGGAAACAATGACAGTTCA Constructing crRNA expression entry clone

CA-crRNA1-R

GGCCTGAACTGTCATTGTTTCCACGTAA Constructing crRNA expression entry clone

CA-crRNA2-F

TAGATACCCTGTGTGAATGGTCAGTAAG Constructing crRNA expression entry clone

CA-crRNA2-R

GGCCCTTACTGACCATTCACACAGGGTA Constructing crRNA expression entry clone

CC-crRNA1-F

TAGATTCCTGAGGAGCAAGAGCCATCAC Constructing crRNA expression entry clone

CC-crRNA1-R

GGCCGTGATGGCTCTTGCTCCTCAGGAA Constructing crRNA expression entry clone

CC-crRNA2-F

TAGATTGTATCTCCGACACCCGGATCAC Constructing crRNA expression entry clone

CC-crRNA2-R

GGCCGTGATCCGGGTGTCGGAGATACAA Constructing crRNA expression entry clone

CG-crRNA1-F

TAGATGATGATGCATCAGGTACTAGAAC Constructing crRNA expression entry clone

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CG-crRNA1-R

GGCCGTTCTAGTACCTGATGCATCATCA Constructing crRNA expression entry clone

CG-crRNA2-F

TAGATGCACCATATGCTTGCTGATCAAA Constructing crRNA expression entry clone

CG-crRNA2-R

GGCCTTTGATCAGCAAGCATATGGTGCA Constructing crRNA expression entry clone

GA-crRNA1-F

TAGATTAATATTGGATCCACCGATTTAA Constructing crRNA expression entry clone

GA-crRNA1-R

GGCCTTAAATCGGTGGATCCAATATTAA Constructing crRNA expression entry clone

GA-crRNA2-F

TAGATGTATTTCTGAATTTCATGCCCAA Constructing crRNA expression entry clone

GA-crRNA2-R

GGCCTTGGGCATGAAATTCAGAAATACA Constructing crRNA expression entry clone

GC-crRNA1-F

TAGATTTCACAATCTTATAGTCACTGAA Constructing crRNA expression entry clone

GC-crRNA1-R

GGCCTTCAGTGACTATAAGATTGTGAAA Constructing crRNA expression entry clone

GC-crRNA2-F

TAGATTGAATGCTCCGGGAGCCGTAGAA Constructing crRNA expression entry clone

GC-crRNA2-R

GGCCTTCTACGGCTCCCGGAGCATTCAA Constructing crRNA expression entry clone

GG-crRNA1-F

TAGATAGCATATGGTTGTAACTTCAGAA Constructing crRNA expression entry clone

GG-crRNA1-R

GGCCTTCTGAAGTTACAACCATATGCTA Constructing crRNA expression entry clone

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Supplemental Figure 1. CRISPR-Cpf1 expression system in this study. (A) crRNA is synthesized as duplexed DNA oligonucleotides and cloned into crRNA expression vectors at Eps3I (BsmBI) sites. These vectors are assembled with Cpf1 and destination vectors to build the final T-DNA vectors for plant transformation. Pre-crRNA with HH (hammerhead ribozyme) and HDV (hepatitis delta virus ribozyme) is self-cleaved to form mature crRNA, which is then combined with Cpf1 for genome editing. (B) Multiple protein sequence alignments of LbCpf1 and FnCpf1 with their variants. Mutations are labeled with red.

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Supplemental Figure 2. Mutations profiles at additional TTV and TTTV PAM sites targeted by FnCpf1. (A) Positions of deletions at six TTTV PAM sites and four TTV PAM sites. (B) Deletion sizes of at six TTTV PAM sites and four TTV PAM sites. Error bars represent standard deviations of two biological replicates.

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Supplemental Figure 3 Genotyping T0 mutant lines by FnCpf1 at TTTV PAM sites with Sanger sequencing.

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pLR453: FnCpf1+OsDEP1-TTTV-crRNA02

WT(OsDEP1): TTCCTTTTCCAGAAAGAGAAGGAGGCACAGATCTTGCCGTCTpLR453-01-01allele 1: TTCCTTTTCCAGAAAGAGAAGG---C------CTTGCCGTCT -9bpallele 2: TTCCTTTTCCAGAAAGAGAAGGAGG----------------- -27bppLR453-01-02 allele 1: TTCCTTTTCCAGAAAGAGAAGG--------------CCGTCT -14bpallele 2: TTCCTTTTCCAGAAAGAGAAGGA------------GCCGTCT -12bppLR453-02-01allele1: TTCCTTTTCCAGAAAGAGAAGGAG----------------CT -16bpallele2: TTCCTTTTCCAGAAAGAGAAGGAG----------TGCCGTCT -10bppLR453-02-02 allele 1: TTCCTTTTCCAGAAAGAGAAGG--------------CCGTCT -14bpallele 2: TTCCTTTTCCAGAAAGAGAAGGAG---------TTGCCGTCT -9bppLR453-02-03allele 1: TTCCTTTTCCAGAAAGAGAAGG--------------CCGTCT -14bpallele 2: TTCCTTTTCCAGAAAGAGAAGGAG---------TTGCCGTCT -9bppLR453-03-01 allele1: TTCCTTTTCCAGAAAGAGAAGGAGGCACAGATCTTGCCGTCT WTallele2: TTCCTTTTCCAGAAAGAGAAGGAGGC-------TTGCCGTCT -7bppLR453-03-02 allele1: TTCCTTTTCCAGAAAGAGAAG-----------CTTGCCGTCT -11bpallele2: TTCCTTTTCCAGAAAGAGAAGGAGGC--------TGCCGTCT -8bppLR453-03-03 allele1: TTCCTTTTCCAGAAAGAGA----------------------- -26bpallele2: TTCCTTTTCCAGAAAGAGAAGGAGG---------TGCCGTCT -9bppLR453-04-01 allele1: TTCCTTTTCCAGAAAGAGAAGGAGGC---------GCCGTCT -9bpallele2: TTCCTTTTCCAGAAAGAGAAGGAGGCA-------TGCCGTCT -7bppLR453-04-02 allele 1: TTCCTTTTCCAGAAAGAGAAGG--------------CCGTCT -14bpallele 2: TTCCTTTTCCAGAAAGAGAAGGAG---------TTGCCGTCT -9bppLR453-04-03 allele 1: TTCCTTTTCCAGAAAGAGAAGG--------------CCGTCT -14bpallele 2: TTCCTTTTCCAGAAAGAGAAGGAG---------TTGCCGTCT -9bppLR453-05-01 allele 1: TTCCTTTTCCAGAAAGAGAAGGAGGC------CTTGCCGTCT -6bpallele 2: TTCCTTTTCCAGAAAGAGAAGGAGGC------CTTGCCGTCT -6bppLR453-05-02allele 1: TTCCTTTTCCAGAAAGAGAAGGAGGC------CTTGCCGTCT -6bpallele 2: TTCCTTTTCCAGAAAGAGAAGGAGGC------CTTGCCGTCT -6bppLR453-06-01allele 1: TTCCTTTTCCAGAAAGAGAAGGAGGC-------TTGCCGTCT -7bpallele 2: TTCCTTTTCCAGAAAGAGAAGGAGGC-C-------------- -17bppLR453-07-01allele 1: TTCCTTTTCCAGAAAGAGAAGGAGGCACAGATCTTGCCGTCT WTallele 2: TTCCTTTTCCAGAAAG----tGAGGC---------------- +1bp/-23bppLR453-08-01allele 1: TTCCTTTTCCAGAAAGAGAAGGAGGC---------GCCGTCT -9bpallele 2: TTCCTTTTCCAGAAAGAGAAGGAGGCA-------TGCCGTCT -7bppLR453-08-02allele 1: TTCCTTTTCCAGAAAGAGAAGGAGGC---------GCCGTCT -9bpallele 2: TTCCTTTTCCAGAAAGAGAAGGAGGCA-------TGCCGTCT -7bppLR453-09-01allele 1: TTCCTTTTCCAGAAAGAGAAGGAG-----GATCTTGCCGTCT -5bpallele 2: TTCCTTTTCCAGAAAGAGAAGGAG-----------------T -17bppLR453-09-02 allele 1: TTCCTTTTCCAGAAAGAGAAGGAGGCACAGATCTTGCCGTCT WTallele 2: TTCCTTTTCCAGAAAGAGAAGGAGGC-----T-TTGCCGTCT -6bppLR453-10-01 allele 1: TTCCTTTTCCAGAAAGAGAAGGAGGC----ATCTTGCCGTCT -5bpallele 2: TTCCTTTTCCAGAAAGAGAAGGAGGC----------CCGTCT -10bppLR453-10-02allele 1: TTCCTTTTCCAGAAAGAGAAGGAG-----GATCTTGCCGTCT -5bpallele 2: TTCCTTTTCCAGAAAGAGAAGGAG----------TGCCGTCT -10bppLR453-11-01allele 1: TTCCTTTT---------------------------------- -37bpallele 2: TTCCTTTT---------------------------------- -37bp pLR453-11-02allele 1: TTCCTTTTCCAGAAAGAGAAGGAGG-------CTTGCCGTCT -7bpallele 2: TTCCTTTTCCAGAAAGAGAAGGAGGAA--------------- +2bp/-27bppLR453-12-01allele 1: TTCCTTTTCCAGAAAGAGAAGGA---------------GTCT -15bpallele 2: TTCCTTTTCCAGAAAGAGAAGGA-----------TG------ -21bppLR453-12-02allele 1: TTCCTTTTCCAGAAAGAGAAGGAGG-------CTTGCCGTCT -7bpallele 2: TTCCTTTTCCAGAAAGAGAAGGAGG-A-A------------- -27bppLR453-12-03allele 1: TTCCTTTTCCAGAAAGAGAAGGAG--------------GTCT -15bpallele 2: TTCCTTTTCCAGAAAGAGAAGGAG------------------ -21bp

pLR464: FnCpf1+OsEPFL9-TTTV-crRNA01

WT(OsEPFL9): ATTTGTTTGAAGAAGGGTTATGGCCAATGCTTGCCCCACATCpLR464-02-03 allele 1: ATTTGTTTGAAGAAGGGTTATGGCCAATGCTTGCCCCACATC WTallele 2: ATTTGTTTGAAGAAGGGTTATGGCCAATG---G--CCACATC -4bppLR464-02-04 allele 1: ATTTGTTTGAAGAAGGGTTATGGCCAATGCTTGCCCCACATC WTallele 2: ATTTGTTTGAAGAAGGGTTATGGCCAATG---G--CCACATC -4bppLR464-02-05 allele 1: ATTTGTTTGAAGAAGGGTTATGGCCAATGCTTGCCCCACATC WTallele 2: ATTTGTTTGAAGAAGGGTTATGGCCAATG---G--CCACATC -4bppLR464-02-06allele 1: ATTTGTTTGAAGAAGGGTTATGGCCAATGCTTGCCCCACATC WTallele 2: ATTTGTTTGAAGAAGGGTTATGG--------TGCCCCACATC -8bppLR464-02-07allele 1: ATTTGTTTGAAGAAGGGTTATGGCC-------GCCCCACATC -7bpallele 2: ATTTGTTTGAAGAAGGGTTATGG---A----TGCCCCACATC -7bppLR464-02-08 allele 1: ATTTGTTTGAAGAAGGGTTATGGCCAATGCTTGCCCCACATC WTallele 2: ATTTGTTTGAAGAAGGGTTATGGC------TTGCCCCACATC -6bpLR464-03-01 allele 1: ATTTGTTTGAAGAAGGGTTATGGCCAATGCTTGCCCCACATC WTallele 2: ATTTGTTTGAAGAAGGGTTATGGC------TTGCCCCACATC -6bppLR464-03-02 allele 1: ATTTGTTTGAAGAAGGGTTATGGCCAATGCTTGCCCCACATC WTallele 2: ATTTGTTTGAAGAAGGGTTATGGC-------TGCCCCACATC -7bppLR464-03-03 allele 1: ATTTGTTTGAAGAAGGGTTATGGCCAATGCTTGCCCCACATC WTallele 2: ATTTGTTTGAAGAAGGGTTATGGCC---------CCCACATC -9bpppLR464-04-02allele 1: ATTTGTTTGAAGAAGGGTTATGGCC-----TTGCCCCACATC -5bpallele 2: ATTTGTTTGAAGAAGGGTTATGGCC---------CCCACATC -9bppLR464-04-03allele 1: ATTTGTTTGAAGAAGGGTTATGG---------GCCCCACATC -9bpallele 2: ATTTGTTTGAAGAAGGGTTATGGC--------GCCCCACATC -8bppLR464-04-04allele 1: ATTTGTTTGAAGAAGGGTTATGGCCAATGCTTGCCCCACATC WTallele 2: ATTTGTTTGAAGAAGGGTTATGGCCAA-----G--CCACATC -7bppLR464-05-01allele 1: ATTTGTTTGAAGAAGGGTTA------------------CATC -18bpallele 2: ATTTGTTTGAAGAAGGGTTATGGCCA------GCCCCACATC -6bppLR464-05-02allele 1: ATTTGTTTGAAGAAGGGTTA------------------CATC -18bpallele 2: ATTTGTTTGAAGAAGGGTTA-----A-----------ACATC -12bppLR464-06-01allele 1: ATTTGTTTGAAGAAGGGTTATGGC------TTGCCCCACATC -6bpallele 2: ATTTGTTTGAAGAAGGGTTATGGC----------CCCACATC -10bppLR464-06-02allele 1: ATTTGTTTGAAGAAGGGTTATGGC------TTGCCCCACATC -6bpallele 2: ATTTGTTTGAAGAAGGGTTATGGC----------CCCACATC -10bppLR464-06-03allele 1: ATTTGTTTGAAGAAGGGTTA------------------CATC -18bpallele 2: ATTTGTTTGAAGAAGGGTTATGGC------TTGCCCCACATC -6bppLR464-07-01 allele 1: ATTTGTTTGAAGAAGGGTTATGG---------GCCCCACATC -9bpallele 2: ATTTGTTTGAAGAAGGGTTATGGCCAA-----GCCCCACATC -5bppLR464-07-02 allele 1: ATTTGTTTGAAGAAGGGTTATGG---------GCCCCACATC -9bpallele 2: ATTTGTTTGAAGAAGGGTTATGGCCAA-----GCCCCACATC -5bppLR464-07-03 allele 1: ATTTGTTTGAAGAAGGGTTATGG---------GCCCCACATC -9bpallele 2: ATTTGTTTGAAGAAGGGTTATGGCCAA-----GCCCCACATC -5bppLR464-08-02 allele 1: ATTTGTTTGAAGAAGGGTTATGGCCAATGCTTGCCCCACATC WTallele 2: -----------------------------------CCACATC -54bppLR464-08-03allele 1: ATTTGTTTGAAGAAGGGTTATGGC--------GCCCCACATC -8bpallele 2: ATTTGTTTGAAGAAGGGTTATGGCC-------GCCCCACATC -7bppLR464-09-01allele 1: ATTTGTTTGAAGAAGGGTTATGGC--ATGCTTGCCCCACATC -2bpallele 2: ATTTGTTTGAAGAAGGGTTATGGC-----CTTGCCCCACATC -5bppLR464-09-02 allele 1: ATTTGTTTGAAGAAGGGTTATGGC--ATGCTTGCCCCACATC -2bpallele 2: ATTTGTTTGAAGAAGGGTTATGGC-----CTTGCCCCACATC -5bppLR464-09-03 allele 1: ATTTGTTTGAAGAAGGGTTATGGC--ATGCTTGCCCCACATC -2bpallele 2: ATTTGTTTGAAGAAGGGTTATGGC-----CTTGCCCCACATC -5bp

Supplemental Figure 4. Genotyping T0 mutant lines by FnCpf1 at TTV PAM sites with Sanger sequencing.

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pLR460: FnCpf1+OsDEP1-TTV-crRNA01

WT(OsDEP1): TCACGTTCCTCAAGGTGAGCGCCCCGCGGCGGCGGCGGCTGpLR460-01-01allele 1: TCACGTTCCTCAAGGTGAGCGCCCCGCGGCGGCGGCGGCTG WTallele 2: TCACGTTCCTCAAGGTGAGCGC--------------GGCTG -14bppLR460-01-02allele 1: TCACGTTCCTCAAGGTGAGCGCCCCGCGGCGGCGGCGGCTG WTallele 2: TCACGTTCCTCAAGGTGAGCG------GGCGGCGGCGGCTG -6bppLR460-01-03allele 1: TCACGTTCCTCAAGGTGAGCGCCCCGCGGCGGCGGCGGCTG WTallele 2: TCACGTTCCTCAAGGTGAGCGCCCCG------CGGCGGCTG -6bppLR460-02-01zallele 1: TCACGTTCCTCAAGGTGAGCGCCCCGCGGCGGCGGCGGCTG WTallele 2: TCACGTTCCTCAAGGTGAGCGCCCCGCGGC---------TG -9bp pLR460-02-02allele 1: TCACGTTCCTCAAGGTGAGCGCC--GCGGCGGCGGCGGCTG -2bpallele 2: TCACGTTCCTCAAGGTGAGCGCC------CGGCGGCGGCTG -6bppLR460-02-03allele 1: TCACGTTCCTCAAGGTGAGCGCCCCGCGGCGGCGGCGGCTG WTallele 2: TCACGTTCCTCAAGGTGAGCGCCC------GGCGGCGGCTG -6bppLR460-03-02allele 1: TCACGTTCCTCAAGGTGAGCGCC-------GGCGGCGGCTG -7bpallele 2: TCACGTTCCTCAAGGTGAGCGCC---------CGGCGGCTG -9bppLR460-03-03allele 1: TCACGTTCCTCAAGGTGAGCGCC-------GGCGGCGGCTG -7bpallele 2: TCACGTTCCTCAAGGTGAGCGCC---------CGGCGGCTG -9bppLR460-04-02allele 1: TCACGTTCCTCAAGGTGAGCGCCCCGCGGCGGCGGCGGCTG WTallele 2: TCACGTTCCTCAAGGTGAGCGCCC---GGCGGCGGCGGCTG -3bppLR460-04-03allele 1: TCACGTTCCTCAAGGTGAGCGCCCCGCGGCGGCGGCGGCTG WTallele 2: TCACGTTCCTCAAGGTGAGCGCCC---GGCGGCGGCCGGTG -3bppLR460-05-02allele 1: TCACGTTCCTCAAGGTGAGCGCC-CGCGGCGGCGGCGGCTG -1bpallele 2: TCACGTTCCTCAAGGTGAGCGCC--------GCGGCGGCTG -8bppLR460-06-03allele 1: TCACGTTCCTCAAGGTGAGCGCCCCGCGGCGGCGGCGGCTG WTallele 2: TCACGTTCCTCAAGGTGAGCGCCCCG-----GCGGCGGCTG -5bppLR460-08-03Allele 1: TCACGTTCCTCAAGGTGAGCGCCCCGCGGCGGCGGCGGCTG WTallele 2: TCACGTTCCTCAAGGTGAGCGCCC------GGCGGCGGCTG -6bppLR460-09-02allele 1: TCACGTTCCTCAAGGTGAGCGCCCCGCGGCGGCGGCGGCTG WTallele 2: TCACGTTCCTCAAGGTGAGCGCCC------GGCGGCGGCTG -6bppLR460-09-03allele 1: TCACGTTCCTCAAGGTGAGCGCCCCGCGGCGGCGGCGGCTG WTallele 2: TCACGTTCCTCAAGGTGAGCGCCC------GGCGGCGGCTG -6bppLR460-10-01allele 1: TCACGTTCCTCAAGGTGAGCGCCCCGCGGCGGCGGCGGCTG WTallele 2: TCACGTTCCTCAAGGTGAGCGCCCCGCGGC---------TG -9bp

pLR465: FnCpf1+OsEPFL9-TTV-crRNA02

WT(OsEPFL9): GCTCGTTGCAATCAAGGGCACCATGGCAGCATCTCAGGTACpLR465-01-01 allele 1: GCTCGTTGCAATCAAGGGCACCATGGCAGCATCTCAGGTAC WTallele 2: GCTCGTTGCAATCAAGGGCACCATG----CATCTCAGGTAC -4bppLR465-01-02 allele 1: GCTCGTTGCAATCAAGGGCACCATGGCAGCATCTCAGGTAC WTallele 2: GCTCGTTGCAATCAAGGGCACCATGGCA---TCTCAGGTAC -3bppLR465-01-03allele 1: GCTCGTTGCAATCAAGGGCACCATGGCAGCATCTCAGGTAC WTallele 2: GCTCGTTGCAATCAAGGGCACCATGGCA---TCTCAGGTAC -3bppLR465-02-01allele 1: GCTCGTTGCAATCA--------------------------- -39bpallele 2: GCTCGTTGCAATCAAGGGCA------------------TAC -18bppLR465-02-02allele 1： GCTCGTTGCAATCAAGGGCACCAT---------CTCAGGTAC -8bpallele 2： GCTCGTTGCAATCAAGGGCACCAT+GGCAGCATCTCAGGTAC +3bp (+3bp:GGT)pLR465-02-03allele 1： GCTCGTTGCAATCAAGGGCACCAT---------CTCAGGTAC -8bpallele 2： GCTCGTTGCAATCAAGGGCACCAT+GGCAGCATCTCAGGTAC +3bp (+3bp:GGT)pLR465-03-01 allele 1: GCTCGTTGCAATCAAGGGCACCATG--------TCAGGTAC -8bpallele 2: GCTCGTTGCAATCAAGGGCACCATGGC-----CTCAGGTAC -5bppLR465-03-02 allele 1: GCTCGTTGCAATCAAGGGCACCATGGCAGCATCTCAGGTAC WTallele 2: GCTCGTTGCAATC--------------------TCAGGTAC -20bppLR465-04-01 allele 1: GCTCGTTGCAATCAAGGGCAC---------ATCTCAGGTAC -9bpallele 2: GCTCGTTGCAATCAAGGGCACCATGG-------------AC -13bppLR465-04-02 allele 1: GCTCGTTGCAATCAAGGGCACCA-----GCATCTCAGGTAC -5bpallele 2: GCTCGTTGCAATCAAGGGCACCATGGCAGCAT-----GTAC -5bppLR465-04-03 allele 1: GCTCGTTGCAATCAAGGGCACCATGGCAGCATCTCAGGTAC WTallele 2: GCTCGTTGCAATCAAGGGCACCA-----G---CT-----AC -13bppLR465-05-01allele 1： GCTCGTTGCAATCAAGGGCACCATGGCAGCATCTCAGGTAC WTallele 2： GCTCGTTGCAATCAAGGGCACCATG------TCTCAGGTAC -6bppLR465-05-02allele 1： GCTCGTTGCAATCAAGGGCACCATGGCAGCATCTCAGGTAC WTallele 2： GCTCGTTGCAATCAAGGGCACCAT--------CTCAGGTAC -8bp

Supplemental Figure 5. RFLP analysis for editing frequencies at VTTV and VTTTV PAM

sites

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Supplemental Figure 6. Mutations profiles at altered PAM sites targeted by LbCpf1 variants. (A) Positions of deletions at 10 TYCV and 3 CCCC PAM sites targeted by LbCpf1-RR and 2 TATG PAM sites targeted by LbCpf1-RVR. (B) Deletion sizes at each target site. Error bars represent standard deviations of two biological replicates.

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Supplemental Figure 7. Mutations profiles at altered PAM sites targeted by FnCpf1 variants. (A) Positions of deletions at 5 TYCV and 1 CCCC PAM sites targeted by FnCpf1-RR and 2 TATG PAM sites targeted by FnCpf1-RVR. (B) Deletion sizes at each target site. Error bars represent standard deviations of two biological replicates.

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Supplemental Figure 8. Genotyping T0 mutant lines edited by Cpf1 variants with Sanger sequencing at six sites.

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Home: Cell Press€¦ · Web viewThe transformed Agrobacterium EHA105 cells were collected and resuspended in liquid AAM-AS medium (OD600=0.1) containing 100μM acetosyringone. After