Supplemental Figure S1. Ten of the Most Prevalent Mutations Generated by Either the Cas9-Grna

Supplemental Figure S1. Ten of the most prevalent mutations generated by either the Cas9-gRNA system (LIG-CR3 gRNA) (A) or the LIG3:4 homing endonuclease (B) identified by deep sequencing of the PCR amplicons across the corresponding target site. gRNA and homing endonuclease target sites are presented in red colored font.

Supplemental Figure S2. Agrobacterium vector for stable integration of the UBI:Cas9 into the maize genome. The same vector was built with temperature regulated promoter, MDH, regulating expression of the Cas9 instead of UBI. These vectors also contain visible marker gene (END2:AmCYAN), which was used for selection of stably transformed callus sectors. Sequence of the Red Fluorescent Protein (DsRED) contained duplicated in a direct orientation 369 bp fragments separated by a 347-bp spacer which contained sequences for recognition and targeting by two gRNAs and LIG3:4 meganuclease. H2B refers to the histone H2B gene promoter.

Supplemental Figure S3. Repair of the RF-FP gene via intramolecular homologous recombination. Embryos with pre-integrated UBI:Cas9 (A) and MDH:Cas9 (B) produced red fluorescing foci 5 days after gRNA (RF-FP-CR1) expression cassette delivery indicating high level of the Cas9 protein expression and activity in the generated transgenic lines. Embryos bombarded only with developmental genes, UBI:ZmODP2 and IN2:ZmWUS, (no gRNA) did not show RF-FP gene repair (C).

Supplemental Figure S4. Constructs used for HDR-mediated targeted gene insertion at the liguleless-1 region using Cas-gRNA system and LIG3:4 meganuclease. A. Donor DNA vector containing a constitutively expressed MoPAT flanked by approximately 1.0 Kb homologous regions (HR1) and (HR2) immediately adjacent to the LIGCas-3 cleavage site. For complete sequence information of the donor DNA, see Supplemental information below. B. Donor DNA vector containing a constitutively expressed MoPAT-DsRED fusion flanked by approximately 1.0 Kb homologous regions (HR1) and (HR2) immediately adjacent to the LIGCas-3 cleavage site retrofitted with UBI:Cas9 and U6:LIG-CR3 gRNA.

Supplemental Table S1. Maize genomic sites targeted by Cas9-gRNA system.

Locus / Target Site Name / Target Site Sequence / PAM
LIG / LIGCas-1 / GTACCGTACGTGCCCCGGCGG / AGG
LIGCas-2 / GGAATTGTACCGTACGTGCCC / CGG
LIGCas-3 / GCGTACGCGTACGTGTG / AGG
Ms26 / MS26Cas-1 / GTACTCCATCCGCCCCATCGAGTA / GGG
MS26Cas-2 / GCACGTACGTCACCATCCCGC / CGG
MS26Cas-3 / GACGTACGTGCCCTACTCGAT / GGG
Ms45 / MS45Cas-1 / GCTGGCCGAGGTCGACTAC / CGG
MS45Cas-2 / GGCCGAGGTCGACTACCGGC / CGG
MS45Cas-3 / GGCGCGAGCTCGTGCTTCAC / CGG
ALS1 and ALS2 / ALSCas-1 / GGTGCCAATCATGCGTCG / CGG
ALSCas-2 / GGTCGCCATCACGGGAC / AGG
ALSCas-3 / GTCGCGGCACCTGTCCCGTGA / TGG

LIG – region approximately 1150 bp upstream of the liguleless-1 gene, Ms26 - male fertility gene 26, Ms45 - male fertility gene 45, ALS1 and ALS2 – acetolactate synthase gene 1 and 2.

Supplemental Table S2. T0 plant analysis by qPCR for mutations at maize target loci of 30 T0 plants produced in the multiplexed Cas9-gRNA experiments.

Targeted Sites / Mutation Analysis by qPCR
LIGCas-3 TS / MS26Cas-2 TS / MS45Cas-2 TS
LIGCas-3 / MS26Cas-2 / null / null
LIGCas-3 / MS26Cas-2 / null / null
LIGCas-3 / MS26Cas-2 / null / null
LIGCas-3 / MS26Cas-2 / null / null
LIGCas-3 / MS26Cas-2 / null / null
LIGCas-3 / MS26Cas-2 / null / null
LIGCas-3 / MS26Cas-2 / null / null
LIGCas-3 / MS26Cas-2 / null / null
LIGCas-3 / MS26Cas-2 / null / null
LIGCas-3 / MS26Cas-2 / null / null
LIGCas-3 / MS26Cas-2 / null / null
LIGCas-3 / MS26Cas-2 / null / null
LIGCas-3 / MS26Cas-2 / null / null
LIGCas-3 / MS26Cas-2 / null / null
LIGCas-3 / MS26Cas-2 / null / null
LIGCas-3 / MS26Cas-2 / null / null
LIGCas-3 / MS26Cas-2 / null / null
LIGCas-3 / MS26Cas-2 / null / null
MS26Cas-2 / MS45Cas-2 / het / null
MS26Cas-2 / MS45Cas-2 / het / null
MS26Cas-2 / MS45Cas-2 / het / null
MS26Cas-2 / MS45Cas-2 / het / null
MS26Cas-2 / MS45Cas-2 / het / null
MS26Cas-2 / MS45Cas-2 / het / null
MS26Cas-2 / MS45Cas-2 / null / null
LIGCas-3 / MS26Cas-2 / MS45Cas-2 / null / null / null
LIGCas-3 / MS26Cas-2 / MS45Cas-2 / null / null / null
LIGCas-3 / MS26Cas-2 / MS45Cas-2 / het / null / null
LIGCas-3 / MS26Cas-2 / MS45Cas-2 / het / null / null
LIGCas-3 / MS26Cas-2 / MS45Cas-2 / null / het / null

Null indicates that both alleles are mutated; het indicates that one allele is mutated and the other wild type. TS, Target site.

Supplemental Table S3. List of primers used in the study.

Target Site / Primer Orientation / Primary PCR Primer Sequence
MS26Cas-1 / Forward / CTACACTCTTTCCCTACACGACGCTCTTCCGATCTAGGACCGGAAGCTCGCCGCGT
Reverse / CAAGCAGAAGACGGCATACGAGCTCTTCCGATCTTCCTGGAGGACGACGTGCTG
MS26Cas-2 / Forward / CTACACTCTTTCCCTACACGACGCTCTTCCGATCTAAGGTCCTGGAGGACGACGTGCTG
Reverse / CAAGCAGAAGACGGCATACGAGCTCTTCCGATCTCCGGAAGCTCGCCGCGT
MS26Cas-3 / Forward / CTACACTCTTTCCCTACACGACGCTCTTCCGATCTTCCTCCGGAAGCTCGCCGCGT
Reverse / CAAGCAGAAGACGGCATACGAGCTCTTCCGATCTTCCTGGAGGACGACGTGCTG
MS26 Meganuclease / Forward / CTACACTCTTTCCCTACACGACGCTCTTCCGATCTTTCCTCCTGGAGGACGACGTGCTG
Reverse / CAAGCAGAAGACGGCATACGAGCTCTTCCGATCTCCGGAAGCTCGCCGCGT
LIGCas-1 / Forward / CTACACTCTTTCCCTACACGACGCTCTTCCGATCTAGGACTGTAACGATTTACGCACCTGCTG
Reverse / CAAGCAGAAGACGGCATACGAGCTCTTCCGATCTGCAAATGAGTAGCAGCGCACGTAT
LIGCas-2 / Forward / CTACACTCTTTCCCTACACGACGCTCTTCCGATCTTCCTCTGTAACGATTTACGCACCTGCTG
Reverse / CAAGCAGAAGACGGCATACGAGCTCTTCCGATCTGCAAATGAGTAGCAGCGCACGTAT
LIGCas-3 / Forward / CTACACTCTTTCCCTACACGACGCTCTTCCGATCTAAGGCGCAAATGAGTAGCAGCGCAC
Reverse / CAAGCAGAAGACGGCATACGAGCTCTTCCGATCTCACCTGCTGGGAATTGTACCGTA
LIG3-4 Meganuclease / Forward / CTACACTCTTTCCCTACACGACGCTCTTCCGATCTCCTTCGCAAATGAGTAGCAGCGCAC
Reverse / CAAGCAGAAGACGGCATACGAGCTCTTCCGATCTCACCTGCTGGGAATTGTACCGTA
MS45Cas-1 / Forward / CTACACTCTTTCCCTACACGACGCTCTTCCGATCTAGGAGGACCCGTTCGGCCTCAGT
Reverse / CAAGCAGAAGACGGCATACGAGCTCTTCCGATCTGCCGGCTGGCATTGTCTCTG
MS45Cas-2 / Forward / CTACACTCTTTCCCTACACGACGCTCTTCCGATCTTCCTGGACCCGTTCGGCCTCAGT
Reverse / CAAGCAGAAGACGGCATACGAGCTCTTCCGATCTGCCGGCTGGCATTGTCTCTG
MS45Cas-3 / Forward / CTACACTCTTTCCCTACACGACGCTCTTCCGATCTGAAGGGACCCGTTCGGCCTCAGT
Reverse / CAAGCAGAAGACGGCATACGAGCTCTTCCGATCTGCCGGCTGGCATTGTCTCTG
ALSCas-1 / Forward / CTACACTCTTTCCCTACACGACGCTCTTCCGATCTAAGGCGACGATGGGCGTCTCCTG
Reverse / CAAGCAGAAGACGGCATACGAGCTCTTCCGATCTGCGTCTGCATCGCCACCTC
ALSCas-2 / Forward / CTACACTCTTTCCCTACACGACGCTCTTCCGATCTTTCCCGACGATGGGCGTCTCCTG
Reverse / CAAGCAGAAGACGGCATACGAGCTCTTCCGATCTGCGTCTGCATCGCCACCTC
ALSCas-3 / Forward / CTACACTCTTTCCCTACACGACGCTCTTCCGATCTGGAACGACGATGGGCGTCTCCTG
Reverse / CAAGCAGAAGACGGCATACGAGCTCTTCCGATCTGCGTCTGCATCGCCACCTC
ALSCas-4 / Forward / CTACACTCTTTCCCTACACGACGCTCTTCCGATCTAAGGGTCTGCATCGCCACCTCCGG
Reverse / CTACACTCTTTCCCTACACGACGCTCTTCCGATCTTTCCGTCTGCATCGCCACCTCCGG
Cas9 specific / Forward / GCGCATATGATCAAGTTCCGCG
Reverse / CGAGGGTGATCTCGGTCTTGAAGAAG
gRNA specific / Forward / TCTGAAAAATGCACCCTCAG
Reverse / CAAGTTGATAACGGACTAGCCTTA
LIG integration junction PCR (HR1) / Forward / GCTCGTGTCCAAGCGTCACTTACGATTAGCT
Reverse / CCCGTTATTGTATGAGGTAATGAC
LIG integration junction PCR (HR2) / Forward / CCATGTCTAACTGTTCATTTATATGATTCTCT
Reverse / GCAGCCGATAGGTTCATCATCTTC
Primers for T7 in vitro transcription
LIG-CR3 gRNA / Forward / GAAATTAATACGACTCACTATAGGCGTACGCGTACGTGTGGTTT
Reverse / AAAAGCACCGACTCGGTGCC
MS26-CR2 gRNA / Forward / GAAATTAATACGACTCACTATAGGCACGTACGTCACCATCCCG
Reverse / AAAAGCACCGACTCGGTGCC
QPCR primer and probes
MS26-Cas2 / MS26 TS F1 / atcctggaggacgacgtgct
Ms26 TS R1 / TTGTACTCCATCCGCCCCAT
MS26 2p1 / 6FAM-CGTCACCATCCCGC
LIG-Cas3 / LIGTS F1 / gatttacgcacctgctggg
LIGTS R1 / CTGAGCTGTATTCCCGCGCA
LIG34 p1 / 6FAM-ATACCTCACACGTACGCG
MS45-Cas2 / MS45TS F2 / TCCTAGTCGCGGACCCGTT
MS45ts R1 / tgcccatgacctccccgta
MS45 1d2 p2 / 6FAM-ACCGGCCGGTAGTC

Supplemental Table S4. Edited ALS2 events recovered using different templates and selection.

DONOR DNA
DNA vector containing 794 bp repair template / ss Oligo 1 / ss Oligo 2
Embryos bombarded / 1000 / 1000 / 1000
# of individual events advanced to Bialophos 4 weeks post-bombardment / 200 / 200 / 200
# of embryos advanced to Chlorosulfron 4 weeks post-bombardment / 800 / 800 / 800
Bialophos resistant events screened / 130 / 130 / 130
# of events producing plants that contained ALS2 edited events / 2a / 3b / 4b
Editing frequency per embroyo (%) / 0.2 / 0.3 / 0.4

a derived from bialophos resistant sectors screened

b derived from chlorosulfron resistant sectors only

Supplemental Table S5. DNA and RNA amounts used in bombardment experiments (single shot).

Mutagenesis / Mgn / gRNA1 / gRNA2 / gRNA3 / gRNA (RNA) / Cas9 / UBI:MoPAT-DsRED / UBI:ODP2 / IN2:WUS
Meganuclease / 50ng / 25ng / 12.5ng / 12.5ng
Single gRNA / 25ng / 80ng / 25ng / 12.5ng / 12.5ng
Duplex gRNA / 20ng / 20ng / 60ng / 25ng / 12.5ng / 12.5ng
Triplex gRNA / 20ng / 20ng / 20ng / 60ng / 25ng / 12.5ng / 12.5ng
Transient gRNA delivery / 100ng / 25ng / 12.5ng / 12.5ng
ALS2 modification / gRNA / Repair DNA / Cas9 / UBI:MoPAT-DsRED / UBI:ODP2 / IN2:WUS
Oligo 1 / 25ng / 40ng / 50ng / 20ng / 6.25ng / 6.25ng
Oligo 2 / 25ng / 40ng / 50ng / 20ng / 6.25ng / 6.25ng
794 bp HR / 20ng / 50ng / 50ng / 20ng / 6.25ng / 6.25ng
Trait gene insertion / Meganuclease vector / gRNA / Cas9 / Repair Template DNA / UBI:ODP2 / IN2:WUS
LIG3:4 Meganuclease1 + donor DNA2
Separate vectors / 50ng / 50ng / 12.5ng / 12.5ng
LIG-CR3 gRNA +
Cas9 +
donor DNA2
Separate vectors / 25ng / 50ng / 50ng / 12.5ng / 12.5ng
LIG-CR3 gRNA +
Cas9 +
donor DNA3
All in a single vector / 125ng / 12.5ng / 12.5ng

Approximate size of vectors:

GuideRNA: 4.4 kb

Cas9: 10.0 kb

Ubi:MoPat: 6.4 kb

Ubi:MoPat-DsRED: 7.1 kb

1LIG3:4 Mgn: 6.0 kb

2LIG repair vector: 8.4 kb

3LIG single vector: 17.5 kb

Ubi:ODP2: 7.8 kb

In2:WUS: 5.6 kb

Supplemental Text S1. Nucleotide sequence and description of the RF-FP repair cassette.

ZmEND2 promoter (green highlight), AmCyan (blue text), CaMV Enhancer (gray highlight), maize Histone promoter (blue highlight), DsRED (red font), spacer (black font) and PinII terminators (red highlight) are shown below. The duplicated direct repeats of the DsRED coding region are underlined. Two gRNA target sites in the spacer region are shown in blue font.

gatccagcttcgcttagtttttagtttttggcagaaaaaatgatcaatgtttcacaaaccaaatatttttataacttttgatgaaagaagatcaccacggtcatatctaggggtggtaacaaattgcgatctaaatgtttcttcataaaaaataaggcttcttaataaattttagttcaaaataaatacgaataaagtctgattctaatctgattcgatccttaaattttataatgcaaaatttagagctcattaccacctctagtcatatgtctagtctgaggtatatccaaaaagccctttctctaaattccacacccaactcagatgtttgcaaataaatactccgactccaaaatgtaggtgaagtgcaactttctccattttatatcaacatttgttattttttgtttaacatttcacactcaaaactaattaataaaatacgtggttgttgaacgtgcgcacatgtctcccttacattatgtttttttatttatgtattattgttgttttcctccgaacaacttgtcaacatatcatcattggtctttaatatttatgaatatggaagcctagttatttacacttggctacacactagttgtagttttgccacttgtctaacatgcaactctagtagttttgccacttgcctggcatgcaactctagtattgacacttgtatagcatataatgccaatacgacacctgccttacatgaaacattatttttgacacttgtataccatgcaacattaccattgacatttgtccatacacattatatcaaatatattgagcgcatgtcacaaactcgatacaaagctggatgaccctccctcaccacatctataaaaacccgagcgctactgtaaatcactcacaacacaacacatatcttttagtaacctttcaataggcgtcccccaagaactagtaaccatggccctgtccaacaagttcatcggcgacgacatgaagatgacctaccacatggacggctgcgtgaacggccactacttcaccgtgaagggcgagggcagcggcaagccctacgagggcacccagacctccaccttcaaggtgaccatggccaacggcggccccctggccttctccttcgacatcctgtccaccgtgttcatgtacggcaaccgctgcttcaccgcctaccccaccagcatgcccgactacttcaagcaggccttccccgacggcatgtcctacgagagaaccttcacctacgaggacggcggcgtggccaccgccagctgggagatcagcctgaagggcaactgcttcgagcacaagtccaccttccacggcgtgaacttccccgccgacggccccgtgatggccaagaagaccaccggctgggacccctccttcgagaagatgaccgtgtgcgacggcatcttgaagggcgacgtgaccgccttcctgatgctgcagggcggcggcaactacagatgccagttccacacctcctacaagaccaagaagcccgtgaccatgccccccaaccacgtggtggagcaccgcatcgccagaaccgacctggacaagggcggcaacagcgtgcagctgaccgagcacgccgtggcccacatcacctccgtggtgcccttctgaagcggcccatggatattcgaacgcgtaggtaccacatggttaacctagacttgtccatcttctggattggccaacttaattaatgtatgaaataaaaggatgcacacatagtgacatgctaatcactataatgtgggcatcaaagttgtgtgttatgtgtaattactagttatctgaataaaagagaaagagatcatccatatttcttatcctaaatgaatgtcacgtgtctttataattctttgatgaaccagatgcatttcattaaccaaatccatatacatataaatattaatcatatataattaatatcaattgggttagcaaaacaaatctagtctaggtgtgttttgcggaattcccatggagtcaaagattcaaatagaggacctaacagaactcgccgtaaagactggcgaacagttcatacagagtctcttacgactcaatgacaagaagaaaatcttcgtcaacatggtggagcacgacacgcttgtctactccaaaaatatcaaagatacagtctcagaagaccaaagggcaattgagacttttcaacaaagggtaatatccggaaacctcctcggattccattgcccagctatctgtcactttattgtgaagatagtggaaaaggaaggtggctcctacaaatgccatcattgcgataaaggaaaggccatcgttgaagatgcctctgccgacagtggtcccaaagatggacccccacccacgaggagcatcgtggaaaaagaagacgttccaaccacgtcttcaaagcaagtggattgatgtgatatctccactgacgtaagggatgacgcacaatcccactaagcttcggccggggcccatcgatctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgccaagctcagatcagcttggggctggtatcgataaatgtttccacatagattttgcatatcataatgatgtttgtcgttccgtatctatgtttcatacaaaatttttacgcatatcgcaacacatgggcacatacctagtgactgtataactctgcatgtatgagtgtatgactatatgatgtagtaactaataagaagggtagacatttgagtgattcttttattcctggacttgtaagacttgacatttctgccttgagtgcgatacatcatatggacaggggttatgcatacactgcttgtttgttgtttatgttctaagagcatctccaacaacgtgacatatgaaaatgccctacaatttaaaaatggttatattttataaaatttagggcataaataaaacatcccgctccaacattaaagccttaaatctattatagggaagcccactatgatatagtatatttgaggcactttagagggtgccctataattttttgaccatttttttatgaaatgagacactattggagtattttttttccgtagagcaccatatttcaatttgagacaccaatttaaggcattgttggagatgttctaaatgttggtttattttgtctgtatcgttgtggttttgatagtggtgcctttgcaatgtacatcttacattgacaataataataggtaaaactctacaaattttttatctaatggactcttgtatgaaacattgtacttgcacacatctgatgtaaacactgcatacttttaacagtgacaagattctgtttcattttagggctagtttgggaaccaaattttattagggtttttattttctaagaaaaagtaatttattttaccttgagaaaatataaattacttgagaaaatagagttccaaactagctcttatctttgtcgaatcctcctctattcaaatgtgacatttctggcacgtgacaactggtgatgttgtagactgtgttaagtaatacgtgtcattattactaaatgccattttagtaaatgttgagtatgtactctactacagtaagtattattggtgtatttacactagacagttggcggcctggcgggtaaagttatcctgtagaaagttgggccaggccaaaaccaaccgccaaaggaaaggccttccggcccgcccacctttgcgcgccgaaggtcagttccttcagtctcctcccgcttcagactctgaccacgtcgacaatccgggccgaaacacatctgcaccgtccacttgcgacagattgaacacaccacttctatccacgtcagcgatccgtggcactagcccttccaccaatcagcccaagttgcccctttcctttaaattcgccgcacccattgctcttctcacggccatagaaatcgaccgagcgaatccctcgcatcgcattcgcagcctttgctgcatcacaccaccgcgaaaccccagcagccgcatctgcaggtcgactctagaggatccatggcctcctccgaggacgtcatcaaggagttcatgcgcttcaaggtgcgcatggagggctccgtgaacggccacgagttcgagatcgagggcgagggcgagggccgcccctacgagggcacccagaccgccaagctgaaggtgaccaagggcggccccctgcccttcgcctgggacatcctgtccccccagttccagtacggctccaaggtgtacgtgaagcaccccgccgacatccccgactacaagaagctgtccttccccgagggcttcaagtgggagcgcgtgatgaacttcgaggacggcggcgtggtgacagtgacccaggactcctccctgcaggacggctccttcatctacaaggtgaagttcatcggcgtgaacttcccctccgacggccccgtaatgcagaagaagactatgggctgggaggcctccaccgagcgcctgtacccccgcgacggcgtgctgaagggcgagatccacaaggccctgaagctgaaggacggcggccacaagcttgctagcccatccacccactcactcactcatatctgtgctgtacgtacgagaatttctcgaccaaccgtcgtgagacctgcccaccggagatcggacgcaagagggtttaggcaagaatgtcgtgcgacagggtgagcgctgactagtatacgtgagagaccttgagatatacctcacacgtacgcgtactttacatgacgtaggacattacgactcaaacagattcacgtcagatttcggagtttctcacgcgtgagagccttggagggcggtatgtatgtcatactatatgttgggatggagggagtgagtgagtgatatgtggctagctaagcttatcgaagggcggccccctgcccttcgcctgggacatcctgtccccccagttccagtacggctccaaggtgtacgtgaagcaccccgccgacatccccgactacaagaagctgtccttccccgagggcttcaagtgggagcgcgtgatgaacttcgaggacggcggcgtggtgacagtgacccaggactcctccctgcaggacggctccttcatctacaaggtgaagttcatcggcgtgaacttcccctccgacggccccgtaatgcagaagaagactatgggctgggaggcctccaccgagcgcctgtacccccgcgacggcgtgctgaagggcgagatccacaaggccctgaagctgaaggacggcggccactacctggtggagttcaagtccatctacatggccaagaagcccgtgcagctgcccggctactactacgtggactccaagctggacatcacctcccacaacgaggactacaccatcgtggagcagtacgagcgcgccgagggccgccaccacctgttcctgtagtcaggatctgagtcgaaacctagacttgtccatcttctggattggccaacttaattaatgtatgaaataaaaggatgcacacatagtgacatgctaatcactataatgtgggcatcaaagttgtgtgttatgtgtaattactagttatctgaataaaagagaaagagatcatccatatttcttatcctaaatgaatgtcacgtgtctttataattctttgatgaaccagatgcatttcattaaccaaatccatatacatataaatattaatcatatataattaatatcaattgggttagcaaaacaaatctagtctaggtgtgttttgcgaatgcggc

Supplemental Text S2. DNA sequences of ALS repair templates

DNA sequences changes relative to wild-type ALS2 gene in repair templates are underlined. Nucleotides modified to change P165 to S are shown in red font.

ALS repair Oligo 1:

AACCTTGTCTCCGCGCTCGCCGACGCGTTGCTCGACTCCGTCCCCATTGTCGCCATCACGGGACAGGTGTCGCGACGCATGATTGGCACCGACGCCTTCCAGGAGACGCCCATCGTCGAGGTCACCC

ALS repair Oligo 2:

AACCTTGTCTCCGCGCTCGCCGACGCGTTGCTGGACTCCGTGCCGATGGTCGCCATCACGGGACAGGTGTCCCGACGCATGATTGGCACCGACGCCTTCCAGGAGACGCCCATCGTCGAGGTCACCC

ALS repair 794 bp fragment:

gacagccgccgcaaccatggccaccgccgccgccgcgtctaccgcgctcactggcgccactaccgctgcgcccaaggcgaggcgccgggcgcacctcctggccacccgccgcgccctcgccgcgcccatcaggtgctcagcggcgtcacccgccatgccgatggctcccccggccaccccgctccggccgtggggccccaccgatccccgcaagggcgccgacatcctcgtcgagtccctcgagcgctgcggcgtccgcgacgtcttcgcctaccccggcggcgcgtccatggagatccaccaggcactcacccgctcccccgtcatcgccaaccacctcttccgccacgagcaaggggaggcctttgcggcctccggctacgcgcgctcctcgggccgcgtcggcgtctgcatcgccacctccggccccggcgccaccaaccttgtctccgcgctcgccgacgcgTtgctcgaCtccgtccccatTgtcgccatcacgggacaggtgTcgcgacgcatgattggcaccgacgccttccaggagacgcccatcgtcgaggtcacccgctccatcaccaagcacaactacctggtcctcgacgtcgacgacatcccccgcgtcgtgcaggaggctttcttcctcgcctcctctggtcgaccggggccggtgcttgtcgacatccccaaggacatccagcagcagatggcggtgcctgtctgggacaagcccatgagtctgcctgggtacattgcgcgccttcccaagccccctgcgactgagttgcttgagcagg

Supplemental Text S3. Nucleotide sequence and description of the donor DNA in the integration experiment.

HR1 and HR2 (blue highlight), UBI:MoPAT:PinII (green highlight), LIGCas-3 target site (red font, underlined), LIG CR-3 gRNA PAM sequence (black font, underlined).

CCCATAGAAAACTGTGTGCTATAATACACCAAAAGGAAAGCAAAGTGAAAAGGAAACTTTGAATAGCCAAGAAGACTCGGAGTGCTTCACGCCTTCACCTATCCCACATAGGTGATGAGCTAAGAGTAAAATGTAGATTCTCTCGAGTACTGAATATTGCCTGCACTTTTCCTTGCAGTAAATACACCTTTAATCCATGACGAGAGTCCACTCTTTGAGTCCGTCTTGAGATTCTTCCATTGATCATACAACATGACCTCGAAGTCCTGATGGAGAACAACTTATATAATTAAAACTACAATACAGAAAGTTCCTGACAATTAAAACCTTTGGTGGTGGCATGCCGTAGGTTAAAAAAAATAGATAATGACAACACAACTGGAGACACGCTCTTTGCCGAGTGCTCACACGTTTGCTGAGAGCGAGCACTCGGCAAATATATGATTTGCCGAATACCACCCTCCTCGGCAAAACAATACACTAGGCAAAAAGGTAGTTTCCCATCACCATGATGCCCGCCGTTAATGTACCTTCTATGCCGAGTATGTTGGCGCTCAGCAAAGAGATCGTTACCGGCGTTTGTTTCACCAAGAGCTCTTTGACGAGTGTGGCACACGACAAAACCTTTTGCCGAGTGTAATTAGTCGTTTGCCAAGTGACTGGTGCAGTTGGCAAAGGAGTCGTTTATTATGTGTGGGCAAAATGATATATGGTGCCAGTTAGGGCTAGCAAATTAAAGGGGGGGGGGGGGGGGTTAGGTTGAAGAAGGTGACGAGTAATAAGGTCTCGGACGGCCGCGCGCATATATATCAGATCCGATCCAATGGCACACGGTGCAAACGAAAAGCACGAAATTTCCACCAGCTTAATTAGGGAGAGAAAAATAGAGCACCAGCTGATGAGTGAATGAATGAGATAGACGGGACACAGAGGGTCCAGCAGGCTAGCCTACTCTGGCCGCCCTAAATAGAAGTCAGTGCCGTGACGACGCGCAAACTTCTTTTGATCGGCTGCGGAAATAATATACTGTAACGATTTACGCACCTGCTGGGAATTGTACCGTACGTGCCCCGGCGGAGGATATATATACCTCACACAAGGGCGAATTGTACTAGTTAGTTAGCTAGTCGGTCCTAGATGCCGTAATCATTAGCTAATCGTAAGTGACGCTTGGACACGAGCGGCTTGAGCTAGGAACCTACGAAGTCATCGGAATCAGCTCAGGTGTACAGAAGTTCCTATACTTTCTGGAGAATAGGAACTTCGGAATAGGAACTTCGTATACGCTAGGGCCGCATTCGCAAAACACACCTAGACTAGATTTGTTTTGCTAACCCAATTGATATTAATTATATATGATTAATATTTATATGTATATGGATTTGGTTAATGAAATGCATCTGGTTCATCAAAGAATTATAAAGACACGTGACATTCATTTAGGATAAGAAATATGGATGATCTCTTTCTCTTTTATTCAGATAACTAGTAATTACACATAACACACAACTTTGATGCCCACATTATAGTGATTAGCATGTCACTATGTGTGCATCCTTTTATTTCATACATTAATTAAGTTGGCCAATCCAGAAGATGGACAAGTCTAGGTTTCGACTCAGATCTGCGTCACCGGGCGCACCGGGCGCGGCGGGGCCGGCAGCTCGAAGTCGCGCTGCCAGAAGCCGACGTCGTGCCAGCCGCCGTGCTTGTAGCCGGCGGCGCGGAGGGTGCCGCGGGCGGTGTAGCCGAGGGCCTCGTGGAGGCGCACGGACGGGTCGTTCGGGAGGCCGATCACGGCCACCACGGACTTGAAGCCCTGGGCCTCCATGCTCTTGAGGAGGTGGGTGTAGAGGGTGGAGCCGAGGCCGAGGCGCTGGTGGCGGTGGGACACGTACACGGTGGACTCCACGGTCCAGTCGTAGGCGTTGCGGGCCTTCCACGGGCCGGCGTAGGCGATGCCGGCCACCACGCCCTCCACCTCGGCCACGAGCCACGGGTAGCGGTCCTGGAGGCGCTCCAGGTCGTCGATCCACTCCTGCGGGGTCTGCGGCTCGGTGCGGAAGTTCACGGTGGAGGTCTCGATGTAGTGGTTCACGATGTCGCACACGGCGGCCATGTCGGCGGCGGTGGCCGGGCGGATCTCGACGGGGCGGCGCTCGGGGGACATGGTGTCGTGTGGATCCCGGTGGATCTGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTTCGCTAGCGAATTGATCCTCTAGAGTCGACCTGCAGAAGTAACACCAAACAACAGGGTGAGCATCGACAAAAGAAACAGTACCAAGCAAATAAATAGCGTATGAAGGCAGGGCTAAAAAAATCCACATATAGCTGCTGCATATGCCATCATCCAAGTATATCAAGATCAAAATAATTATAAAACATACTTGTTTATTATAATAGATAGGTACTCAAGGTTAGAGCATATGAATAGATGCTGCATATGCCATCATGTATATGCATCAGTAAAACCCACATCAACATGTATACCTATCCTAGATCGATATTTCCATCCATCTTAAACTCGTAACTATGAAGATGTATGACACACACATACAGTTCCAAAATTAATAAATACACCAGGTAGTTTGAAACAGTATTCTACTCCGATCTAGAACGAATGAACGACCGCCCAACCACACCACATCATCACAACCAAGCGAACAAAAAGCATCTCTGTATATGCATCAGTAAAACCCGCATCAACATGTATACCTATCCTAGATCGATATTTCCATCCATCATCTTCAATTCGTAACTATGAATATGTATGGCACACACATACAGATCCAAAATTAATAAATCCACCAGGTAGTTTGAAACAGAATTCTACTCCGATCTAGAACGACCGCCCAACCAGACCACATCATCACAACCAAGACAAAAAAAAGCATGAAAAGATGACCCGACAAACAAGTGCACGGCATATATTGAAATAAAGGAAAAGGGCAAACCAAACCCTATGCAACGAAACAAAAAAAATCATGAAATCGATCCCGTCTGCGGAACGGCTAGAGCCATCCCAGGATTCCCCAAAGAGAAACACTGGCAAGTTAGCAATCAGAACGTGTCTGACGTACAGGTCGCATCCGTGTACGAACGCTAGCAGCACGGATCTAACACAAACACGGATCTAACACAAACATGAACAGAAGTAGAACTACCGGGCCCTAACCATGCATGGACCGGAACGCCGATCTAGAGAAGGTAGAGAGGGGGGGGGGGGAGGACGAGCGGCGTACCTTGAAGCGGAGGTGCCGACGGGTGGATTTGGGGGAGATCTGGTTGTGTGTGTGTGCGCTCCGAACAACACGAGGTTGGGGAAAGAGGGTGTGGAGGGGGTGTCTATTTATTACGGCGGGCGAGGAAGGGAAAGCGAAGGAGCGGTGGGAAAGGAATCCCCCGTAGCTGCCGGTGCCGTGAGAGGAGGAGGAGGCCGCCTGCCGTGCCGGCTCACGTCTGCCGCTCCGCCACGCAATTTCTGGATGCCGACAGCGGAGCAAGTCCAACGGTGGAGCGGAACTCTCGAGAGGGGTCCAGAGGCAGCGACAGAGATGCCGTGCCGTCTGCTTCGCTTGGCCCGACGCGACGCTGCTGGTTCGCTGGTTGGTGTCCGTTAGACTCGTCGACGGCGTTTAACAGGCTGGCATTATCTACTCGAAACAAGAAAAATGTTTCCTTAGTTTTTTTAATTTCTTAAAGGGTATTTGTTTAATTTTTAGTCACTTTATTTTATTCTATTTTATATCTAAATTATTAAATAAAAAAACTAAAATAGAGTTTTAGTTTTCTTAATTTAGAGGCTAAAATAGAATAAAATAGATGTACTAAAAAAATTAGTCTATAAAAACCATTAACCCTAAACCCTAAATGGATGTACTAATAAAATGGATGAAGTATTATATAGGTGAAGCTATTTGCAAAAAAAAAGGAGAACACATGCACACTAAAAAGATAAAACTGTAGAGTCCTGTTGTCAAAATACTCAATTGTCCTTTAGACCATGTCTAACTGTTCATTTATATGATTCTCTAAAACACTGATATTATTGTAGTACTATAGATTATATTATTCGTAGAGTAAAGTTTAAATATATGTATAAAGATAGATAAACTGCACTTCAAACAAGTGTGACAAAAAAAATATGTGGTAATTTTTTATAACTTAGACATGCAATGCTCATTATCTCTAGAGAGGGGCACGACCGGGTCACGCTGCACTGCAGGCTAGCGGCGAATTCGCCCTTGTACGCGTACGCGTATATATACGTGCGCTGCTACTCATTTGCGCGGGAATACAGCTCAGTCTGCTGTGCGCTGCAGGATGTACATACATACATGCGCAGGTGCAAAGTCTACGCGCGCGGGCAATGCAAGCCCCTGGCGTAGTTGGGCCATGACTGAGATCACGCCTCATGGTCATGGAACGAAACACCGCGTCCGGCCGGGCTGCCCCTGGCGTCACGCGGGAGGCAGCTGCTAGCGTTAGCGTACGTACCCACCGTCTCGTACACACCACCGCAGGGAGAGAGAAGAGCGATGCAATGCACATGTACAGCATCCGCATCATGCATAGATACTCATATCTTCAAGGCCACACATGCAGCAGTGTCGTACGCTACGTTGTTTCAACGGAGGAGGAGGATACATACATAGACACCCACAGCCAGCCTAGCATATAGCAGATAGCATACGGACTCCCGGGTGAGGAAAAATGGAGGGCGAACCAAACCAACCACAAAGAAGCAGCAGCAGCAGCAGCAGCAGCTGCGGCTGCTATCACCACTCACCAACTCCAATTAAAGATCTCTCTCTCTCTCTCTACTGGCCGGCCCTGTCAGTGCCAGCGCCCGGTTTGTTGCTAGCTGAGCTGCGGGCGTCGCTCTTAGATATAGCCCAAAACTCACTCCACCACCACTCGTTCCATGGAACCCTAGACCAAAAGTACTCGCGCTCTCGGCCCTCGCTCTCGCCCTCTCCCTCTCCGCAGCAAAAGAGATCCGGCCGGCCGAGAAGGGCGCGCGCTAGCTGCCCGGCTACTAGCTGGCGCCCGCCCGCGCATATATCTGTGTCATCGCCATCACCCACACCATGGCCCGGCCGGCCAACACCGCCGTATTAGCTCTGTCTGTCGCTCGTCCACCTGCGACCGACTGAGCGATCGATCTCCACCGAGCTCTCCGCTAAGCGCTGTCCTTGCCGCCGTCCTCCCCTCCGTCCCCTACGCATCCATTTCCGTGTGCTCGTGTGTGCGCGCGCGGGCACTCCTGCTCCTGCTCCCTCCGGCCCCTCCTCCCCTCCCAGGCTCCCAGCTAGCCGCGCCCGCCCGCGCGACCTGCACCTGCACAGATCGGGCGGCCGGGCCGACCGATCGATCGAGAT

Supplemental Text S4. Nucleotide sequence of maize-optimized Cas9.

atggcaccgaagaagaagcgcaaggtgatggacaagaagtacagcatcggcctcgacatcggcaccaactcggtgggctgggccgtcatcacggacgaatataaggtcccgtcgaagaagttcaaggtcctcggcaatacagaccgccacagcatcaagaaaaacttgatcggcgccctcctgttcgatagcggcgagaccgcggaggcgaccaggctcaagaggaccgccaggagacggtacactaggcgcaagaacaggatctgctacctgcaggagatcttcagcaacgagatggcgaaggtggacgactccttcttccaccgcctggaggaatcattcctggtggaggaggacaagaagcatgagcggcacccaatcttcggcaacatcgtcgacgaggtaagtttctgcttctacctttgatatatatataataattatcattaattagtagtaatataatatttcaaatatttttttcaaaataaaagaatgtagtatatagcaattgcttttctgtagtttataagtgtgtatattttaatttataacttttctaatatatgaccaaaacatggtgatgtgcaggtggcctaccacgagaagtacccgacaatctaccacctccggaagaaactggtggacagcacagacaaggcggacctccggctcatctaccttgccctcgcgcatatgatcaagttccgcggccacttcctcatcgagggcgacctgaacccggacaactccgacgtggacaagctgttcatccagctcgtgcagacgtacaatcaactgttcgaggagaaccccataaacgctagcggcgtggacgccaaggccatcctctcggccaggctctcgaaatcaagaaggctggagaaccttatcgcgcagttgccaggcgaaaagaagaacggcctcttcggcaaccttattgcgctcagcctcggcctgacgccgaacttcaaatcaaacttcgacctcgcggaggacgccaagctccagctctcaaaggacacctacgacgacgacctcgacaacctcctggcccagataggagaccagtacgcggacctcttcctcgccgccaagaacctctccgacgctatcctgctcagcgacatccttcgggtcaacaccgaaattaccaaggcaccgctgtccgccagcatgattaaacgctacgacgagcaccatcaggacctcacgctgctcaaggcactcgtccgccagcagctccccgagaagtacaaggagatcttcttcgaccaatcaaaaaacggctacgcgggatatatcgacggcggtgccagccaggaagagttctacaagttcatcaaaccaatcctggagaagatggacggcaccgaggagttgctggtcaagctcaacagggaggacctcctcaggaagcagaggaccttcgacaacggctccatcccgcatcagatccacctgggcgaactgcatgccatcctgcggcgccaggaggacttctacccgttcctgaaggataaccgggagaagatcgagaagatcttgacgttccgcatcccatactacgtgggcccgctggctcgcggcaactcccggttcgcctggatgacccggaagtcggaggagaccatcacaccctggaactttgaggaggtggtcgataagggcgctagcgctcagagcttcatcgagcgcatgaccaacttcgataaaaacctgcccaatgaaaaagtcctccccaagcactcgctgctctacgagtacttcaccgtgtacaacgagctcaccaaggtcaaatacgtcaccgagggcatgcggaagccggcgttcctgagcggcgagcagaagaaggcgatagtggacctcctcttcaagaccaacaggaaggtgaccgtgaagcaattaaaagaggactacttcaagaaaatagagtgcttcgactccgtggagatctcgggcgtggaggatcggttcaacgcctcactcggcacgtatcacgacctcctcaagatcattaaagacaaggacttcctcgacaacgaggagaacgaggacatcctcgaggacatcgtcctcaccctgaccctgttcgaggaccgcgaaatgatcgaggagaggctgaagacctacgcgcacctgttcgacgacaaggtcatgaaacagctcaagaggcgccgctacactggttggggaaggctgtcccgcaagctcattaatggcatcagggacaagcagagcggcaagaccatcctggacttcctcaagtccgacgggttcgccaaccgcaacttcatgcagctcattcacgacgactcgctcacgttcaaggaagacatccagaaggcacaggtgagcgggcagggtgactccctccacgaacacatcgccaacctggccggctcgccggccattaaaaagggcatcctgcagacggtcaaggtcgtcgacgagctcgtgaaggtgatgggccggcacaagcccgaaaatatcgtcatagagatggccagggagaaccagaccacccaaaaagggcagaagaactcgcgcgagcggatgaaacggatcgaggagggcattaaagagctcgggtcccagatcctgaaggagcaccccgtggaaaatacccagctccagaatgaaaagctctacctctactacctgcagaacggccgcgacatgtacgtggaccaggagctggacattaatcggctatcggactacgacgtcgaccacatcgtgccgcagtcgttcctcaaggacgatagcatcgacaacaaggtgctcacccggtcggataaaaatcggggcaagagcgacaacgtgcccagcgaggaggtcgtgaagaagatgaaaaactactggcgccagctcctcaacgcgaaactgatcacccagcgcaagttcgacaacctgacgaaggcggaacgcggtggcttgagcgaactcgataaggcgggcttcataaaaaggcagctggtcgagacgcgccagatcacgaagcatgtcgcccagatcctggacagccgcatgaatactaagtacgatgaaaacgacaagctgatccgggaggtgaaggtgatcacgctgaagtccaagctcgtgtcggacttccgcaaggacttccagttctacaaggtccgcgagatcaacaactaccaccacgcccacgacgcctacctgaatgcggtggtcgggaccgccctgatcaagaagtacccgaagctggagtcggagttcgtgtacggcgactacaaggtctacgacgtgcgcaaaatgatcgccaagtccgagcaggagatcggcaaggccacggcaaaatacttcttctactcgaacatcatgaacttcttcaagaccgagatcaccctcgcgaacggcgagatccgcaagcgcccgctcatcgaaaccaacggcgagacgggcgagatcgtctgggataagggccgggatttcgcgacggtccgcaaggtgctctccatgccgcaagtcaatatcgtgaaaaagacggaggtccagacgggcgggttcagcaaggagtccatcctcccgaagcgcaactccgacaagctcatcgcgaggaagaaggattgggacccgaaaaaatatggcggcttcgacagcccgaccgtcgcatacagcgtcctcgtcgtggcgaaggtggagaagggcaagtcaaagaagctcaagtccgtgaaggagctgctcgggatcacgattatggagcggtcctccttcgagaagaacccgatcgacttcctagaggccaagggatataaggaggtcaagaaggacctgattattaaactgccgaagtactcgctcttcgagctggaaaacggccgcaagaggatgctcgcctccgcaggcgagttgcagaagggcaacgagctcgccctcccgagcaaatacgtcaatttcctgtacctcgctagccactatgaaaagctcaagggcagcccggaggacaacgagcagaagcagctcttcgtggagcagcacaagcattacctggacgagatcatcgagcagatcagcgagttctcgaagcgggtgatcctcgccgacgcgaacctggacaaggtgctgtcggcatataacaagcaccgcgacaaaccaatacgcgagcaggccgaaaatatcatccacctcttcaccctcaccaacctcggcgctccggcagccttcaagtacttcgacaccacgattgaccggaagcggtacacgagcacgaaggaggtgctcgatgcgacgctgatccaccagagcatcacagggctctatgaaacacgcatcgacctgagccagctgggcggagacaagagaccacgggaccgccacgatggcgagctgggaggccgcaagcgggcaaggtag

Supplemental Text S5. Nucleotide sequence of LIG-CR3 gRNA expression cassette.

Expression cassette containing ZmPolIII promoter, LIG-CR3 spacer (underlined), single guideRNA (red font) and PolIII terminator.

tgagagtacaatgatgaacctagattaatcaatgccaaagtctgaaaaatgcaccctcagtctatgatccagaaaatcaagattgcttgaggccctgttcggttgttccggattagagccccggattaattcctagccggattacttctctaatttatatagattttgatgagctggaatgaatcctggcttattccggtacaaccgaacaggccctgaaggataccagtaatcgctgagctaaattggcatgctgtcagagtgtcagtattgcagcaaggtagtgagataaccggcatcatggtgccagtttgatggcaccattagggttagagatggtggccatgggcgcatgtcctggccaactttgtatgatatatggcagggtgaataggaaagtaaaattgtattgtaaaaagggatttcttctgtttgttagcgcatgtacaaggaatgcaagttttgagcgagggggcatcaaagatctggctgtgtttccagctgtttttgttagccccatcgaatccttgacataatgatcccgcttaaataagcaacctcgcttgtatagttccttgtgctctaacacacgatgatgataagtcgtaaaatagtggtgtccaaagaatttccaggcccagttgtaaaagctaaaatgctattcgaatttctactagcagtaagtcgtgtttagaaattatttttttatataccttttttccttctatgtacagtaggacacagtgtcagcgccgcgttgacggagaatatttgcaaaaaagtaaaagagaaagtcatagcggcgtatgtgccaaaaacttcgtcacagagagggccataagaaacatggcccacggcccaatacgaagcaccgcgacgaagcccaaacagcagtccgtaggtggagcaaagcgctgggtaatacgcaaacgttttgtcccaccttgactaatcacaagagtggagcgtaccttataaaccgagccgcaagcaccgaattgcgtacgcgtacgtgtggttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcttttttttt