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.

5’-GCGUACGCGUACGUGUG - gRNA-3’

cleavage site

CACGTATATATACGCGTACGCGTACGTGTGAGGTATATATATCCTCCGCCGGGGCACGTAC Count CACGTATATATACGCGTACGCGTACGTTGTGAGGTATATATATCCTCCGCCGGGGCACGTAC 16861 CACGTATATATACGCGTACGCGTACG-GTGAGGTATATATATCCTCCGCCGGGGCACGTAC 3684 CACGTATATATACGCGTACGCGTAC-TGTGAGGTATATATATCCTCCGCCGGGGCACGTAC 2263 CACGTATATATACGCGTACGCGTACG—-TGAGGTATATATATCCTCCGCCGGGGCACGTAC 2132 CACGTATATATA-----------------------------TCCTCCGCCGGGGCACGTAC 1181 CACGTATATATACG------------------------------------------CGTAC 848 CACGTATATATACGCGTACGCGT----GTGAGGTATATATATCCTCCGCCGGGGCACGTAC 327 CACGTATATATACGCGTACGC----------------------------CGGGGCACGTAC 263 CACGTATATAT-------------------------------CCTCCGCCGGGGCACGTAC 227 CACGTATATATACGCGTACGCGTA—-TGTGAGGTATATATATCCTCCGCCGGGGCACGTAC 209

A

B cleavage site

CACGTATATATACGCGTACGCGTACGTGTGAGGTATATATATCCTCCGCCGGGGCACGTAC Count CACGTATATATACGCGTACGCGTACGTG--AGGTATATATATCCTCCGCCGGGGCACGTAC 358 CACGTATATATA-----------------------------TCCTCCGCCGGGGCACGTAC 241 CACGTATATATACGCGTACGCGT--------------------------------ACGTAC 150 CACGTATATATACG------------------------------------------CGTAC 143 CACGTATATATACGCGTA----------------------------CGCCGGGGCACGTAC 97 CACGTATATATACGCGT------------GAGGTATATATATCCTCCGCCGGGGCACGTAC 52 CACGTATATATACGCGTACGCGT----GTGAGGTATATATATCCTCCGCCGGGGCACGTAC 50 CACGTATATATACGCGT-------------------------CCTCCGCCGGGGCACGTAC 46 CACGTATATATACGCGTACGCGTACGTGT--GGTATATATATCCTCCGCCGGGGCACGTAC 42 CACGTATATATACGCGTACGCGTACG-----GGTATATATATCCTCCGCCGGGGCACGTAC 32

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.

RFF FPspacerAmCYAN H2B pro

35S ENHEND2 pro

UBI Cas9 LBRB

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).

1 mm

CBA

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.

UBI:MoPAT HR2HR1

UBI:MoPAT-DsRED HR2HR1U6:LIG-CR3UBI:Cas9

A

B

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

Locus Target Site Name Target Site Sequence PAM

LIGLIGCas-1 GTACCGTACGTGCCCCGGCGG AGGLIGCas-2 GGAATTGTACCGTACGTGCCC CGGLIGCas-3 GCGTACGCGTACGTGTG AGG

Ms26MS26Cas-1 GTACTCCATCCGCCCCATCGAGTA GGGMS26Cas-2 GCACGTACGTCACCATCCCGC CGGMS26Cas-3 GACGTACGTGCCCTACTCGAT GGG

Ms45MS45Cas-1 GCTGGCCGAGGTCGACTAC CGGMS45Cas-2 GGCCGAGGTCGACTACCGGC CGGMS45Cas-3 GGCGCGAGCTCGTGCTTCAC CGG

ALS1 and ALS2

ALSCas-1 GGTGCCAATCATGCGTCG CGGALSCas-2 GGTCGCCATCACGGGAC AGGALSCas-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 qPCRLIGCas-3 TS MS26Cas-2 TS MS45Cas-2 TS

LIGCas-3 / MS26Cas-2 null nullLIGCas-3 / MS26Cas-2 null nullLIGCas-3 / MS26Cas-2 null nullLIGCas-3 / MS26Cas-2 null nullLIGCas-3 / MS26Cas-2 null nullLIGCas-3 / MS26Cas-2 null nullLIGCas-3 / MS26Cas-2 null nullLIGCas-3 / MS26Cas-2 null nullLIGCas-3 / MS26Cas-2 null nullLIGCas-3 / MS26Cas-2 null nullLIGCas-3 / MS26Cas-2 null nullLIGCas-3 / MS26Cas-2 null nullLIGCas-3 / MS26Cas-2 null nullLIGCas-3 / MS26Cas-2 null nullLIGCas-3 / MS26Cas-2 null nullLIGCas-3 / MS26Cas-2 null nullLIGCas-3 / MS26Cas-2 null nullLIGCas-3 / MS26Cas-2 null null

MS26Cas-2 / MS45Cas-2 het nullMS26Cas-2 / MS45Cas-2 het nullMS26Cas-2 / MS45Cas-2 het nullMS26Cas-2 / MS45Cas-2 het nullMS26Cas-2 / MS45Cas-2 het nullMS26Cas-2 / MS45Cas-2 het nullMS26Cas-2 / MS45Cas-2 null null

LIGCas-3 / MS26Cas-2 / MS45Cas-2 null null nullLIGCas-3 / MS26Cas-2 / MS45Cas-2 null null nullLIGCas-3 / MS26Cas-2 / MS45Cas-2 het null nullLIGCas-3 / MS26Cas-2 / MS45Cas-2 het null nullLIGCas-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-1Forward CTACACTCTTTCCCTACACGACGCTCTTCCGATCTAGGACCGGAAGCTCGCCGCGTReverse CAAGCAGAAGACGGCATACGAGCTCTTCCGATCTTCCTGGAGGACGACGTGCTG

MS26Cas-2Forward CTACACTCTTTCCCTACACGACGCTCTTCCGATCTAAGGTCCTGGAGGACGACGTGCTGReverse CAAGCAGAAGACGGCATACGAGCTCTTCCGATCTCCGGAAGCTCGCCGCGT

MS26Cas-3Forward CTACACTCTTTCCCTACACGACGCTCTTCCGATCTTCCTCCGGAAGCTCGCCGCGTReverse CAAGCAGAAGACGGCATACGAGCTCTTCCGATCTTCCTGGAGGACGACGTGCTG

MS26 MeganucleaseForward CTACACTCTTTCCCTACACGACGCTCTTCCGATCTTTCCTCCTGGAGGACGACGTGCTGReverse CAAGCAGAAGACGGCATACGAGCTCTTCCGATCTCCGGAAGCTCGCCGCGT

LIGCas-1Forward CTACACTCTTTCCCTACACGACGCTCTTCCGATCTAGGACTGTAACGATTTACGCACCTGCTGReverse CAAGCAGAAGACGGCATACGAGCTCTTCCGATCTGCAAATGAGTAGCAGCGCACGTAT

LIGCas-2Forward CTACACTCTTTCCCTACACGACGCTCTTCCGATCTTCCTCTGTAACGATTTACGCACCTGCTGReverse CAAGCAGAAGACGGCATACGAGCTCTTCCGATCTGCAAATGAGTAGCAGCGCACGTAT

LIGCas-3Forward CTACACTCTTTCCCTACACGACGCTCTTCCGATCTAAGGCGCAAATGAGTAGCAGCGCACReverse CAAGCAGAAGACGGCATACGAGCTCTTCCGATCTCACCTGCTGGGAATTGTACCGTA

LIG3-4 MeganucleaseForward CTACACTCTTTCCCTACACGACGCTCTTCCGATCTCCTTCGCAAATGAGTAGCAGCGCACReverse CAAGCAGAAGACGGCATACGAGCTCTTCCGATCTCACCTGCTGGGAATTGTACCGTA

MS45Cas-1Forward CTACACTCTTTCCCTACACGACGCTCTTCCGATCTAGGAGGACCCGTTCGGCCTCAGTReverse CAAGCAGAAGACGGCATACGAGCTCTTCCGATCTGCCGGCTGGCATTGTCTCTG

MS45Cas-2Forward CTACACTCTTTCCCTACACGACGCTCTTCCGATCTTCCTGGACCCGTTCGGCCTCAGTReverse CAAGCAGAAGACGGCATACGAGCTCTTCCGATCTGCCGGCTGGCATTGTCTCTG

MS45Cas-3Forward CTACACTCTTTCCCTACACGACGCTCTTCCGATCTGAAGGGACCCGTTCGGCCTCAGTReverse CAAGCAGAAGACGGCATACGAGCTCTTCCGATCTGCCGGCTGGCATTGTCTCTG

ALSCas-1Forward CTACACTCTTTCCCTACACGACGCTCTTCCGATCTAAGGCGACGATGGGCGTCTCCTGReverse CAAGCAGAAGACGGCATACGAGCTCTTCCGATCTGCGTCTGCATCGCCACCTC

ALSCas-2Forward CTACACTCTTTCCCTACACGACGCTCTTCCGATCTTTCCCGACGATGGGCGTCTCCTGReverse CAAGCAGAAGACGGCATACGAGCTCTTCCGATCTGCGTCTGCATCGCCACCTC

ALSCas-3Forward CTACACTCTTTCCCTACACGACGCTCTTCCGATCTGGAACGACGATGGGCGTCTCCTGReverse CAAGCAGAAGACGGCATACGAGCTCTTCCGATCTGCGTCTGCATCGCCACCTC

ALSCas-4Forward CTACACTCTTTCCCTACACGACGCTCTTCCGATCTAAGGGTCTGCATCGCCACCTCCGGReverse CTACACTCTTTCCCTACACGACGCTCTTCCGATCTTTCCGTCTGCATCGCCACCTCCGG

Cas9 specificForward GCGCATATGATCAAGTTCCGCGReverse CGAGGGTGATCTCGGTCTTGAAGAAG

gRNA specificForward TCTGAAAAATGCACCCTCAGReverse CAAGTTGATAACGGACTAGCCTTA

LIG integration junction PCR (HR1)

Forward GCTCGTGTCCAAGCGTCACTTACGATTAGCTReverse CCCGTTATTGTATGAGGTAATGAC

LIG integration junction PCR (HR2)

Forward CCATGTCTAACTGTTCATTTATATGATTCTCTReverse GCAGCCGATAGGTTCATCATCTTC

Primers for T7 in vitro transcription

LIG-CR3 gRNAForward GAAATTAATACGACTCACTATAGGCGTACGCGTACGTGTGGTTTReverse AAAAGCACCGACTCGGTGCC

MS26-CR2 gRNAForward GAAATTAATACGACTCACTATAGGCACGTACGTCACCATCCCGReverse AAAAGCACCGACTCGGTGCC

QPCR primer and probes

MS26-Cas2MS26 TS F1 ATCCTGGAGGACGACGTGCTMs26 TS R1 TTGTACTCCATCCGCCCCATMS26 2p1 6FAM-CGTCACCATCCCGC

LIG-Cas3LIGTS F1 GATTTACGCACCTGCTGGGLIGTS R1 CTGAGCTGTATTCCCGCGCALIG34 p1 6FAM-ATACCTCACACGTACGCG

MS45-Cas2MS45TS F2 TCCTAGTCGCGGACCCGTTMS45TS R1 TGCCCATGACCTCCCCGTAMS45 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-

bombardment200 200 200

# of embryos advanced to Chlorosulfron 4 weeks post-

bombardment800 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

DNAUBI: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 kbCas9: 10.0 kbUbi:MoPat: 6.4 kbUbi:MoPat-DsRED: 7.1 kb1LIG3:4 Mgn: 6.0 kb2LIG repair vector: 8.4 kb3LIG single vector: 17.5 kbUbi:ODP2: 7.8 kbIn2: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).

CCCATAGAAAACTGTGTGCTATAATACACCAAAAGGAAAGCAAAGTGAAAAGGAAACTTTGAATAGCCAAGAAGACTCGGAGTGCTTCACGCCTTCACCTATCCCACATAGGTGATGAGCTAAGAGTAAAATGTAGATTCTCTCGAGTACTGAATATTGCCTGCACTTTTCCTTGCAGTAAATACACCTTTAATCCATGACGAGAGTCCACTCTTTGAGTCCGTCTTGAGATTCTTCCATTGATCATACAACATGACCTCGAAGTCCTGATGGAGAACAACTTATATAATTAAAACTACAATACAGAAAGTTCCTGACAATTAAAACCTTTGGTGGTGGCATGCCGTAGGTTAAAAAAAATAGATAATGACAACACAACTGGAGACACGCTCTTTGCCGAGTGCTCACACGTTTGCTGAGAGCGAGCACTCGGCAAATATATGATTTGCCGAATACCACCCTCCTCGGCAAAACAATACACTAGGCAAAAAGGTAGTTTCCCATCACCATGATGCCCGCCGTTAATGTACCTTCTATGCCGAGTATGTTGGCGCTCAGCAAAGAGATCGTTACCGGCGTTTGTTTCACCAAGAGCTCTTTGACGAGTGTGGCACACGACAAAACCTTTTGCCGAGTGTAATTAGTCGTTTGCCAAGTGACTGGTGCAGTTGGCAAAGGAGTCGTTTATTATGTGTGGGCAAAATGATATATGGTGCCAGTTAGGGCTAGCAAATTAAAGGGGGGGGGGGGGGGGTTAGGTTGAAGAAGGTGACGAGTAATAAGGTCTCGGACGGCCGCGCGCATATATATCAGATCCGATCCAATGGCACACGGTGCAAACGAAAAGCACGAAATTTCCACCAGCTTAATTAGGGAGAGAAAAATAGAGCACCAGCTGATGAGTGAATGAATGAGATAGACGGGACACAGAGGGTCCAGCAGGCTAGCCTACTCTGGCCGCCCTAAATAGAAGTCAGTGCCGTGACGACGCGCAAACTTCTTTTGATCGGCTGCGGAAATAATATACTGTAACGATTTACGCACCTGCTGGGAATTGTACCGTACGTGCCCCGGCGGAGGATATATATACCT CACAC AAGGGCGAATTGTACTAGTTAGTTAGCTAGTCGGTCCTAGATGCCGTAATCATTAGCTAATCGTAAGTGACGCTTGGACACGAGCGGCTTGAGCTAGGAACCTACGAAGTCATCGGAATCAGCTCAGGTGTACAGAAGTTCCTATACTTTCTGGAGAATAGGAACTTCGGAATAGGAACTTCGTATACGCTAGGGCCGCATTCGCAAAACACACCTAGACTAGATTTGTTTTGCTAACCCAATTGATATTAATTATATATGATTAATATTTATATGTATATGGATTTGGTTAATGAAATGCATCTGGTTCATCAAAGAATTATAAAGACACGTGACATTCATTTAGGATAAGAAATATGGATGATCTCTTTCTCTTTTATTCAGATAACTAGTAATTACACATAACACACAACTTTGATGCCCACATTATAGTGATTAGCATGTCACTATGTGTGCATCCTTTTATTTCATACATTAATTAAGTTGGCCAATCCAGAAGATGGACAAGTCTAGGTTTCGACTCAGATCTGCGTCACCGGGCGCACCGGGCGCGGCGGGGCCGGCAGCTCGAAGTCGCGCTGCCAGAAGCCGACGTCGTGCCAGCCGCCGTGCTTGTAGCCGGCGGCGCGGAGGGTGCCGCGGGCGGTGTAGCCGAGGGCCTCGTGGAGGCGCACGGACGGGTCGTTCGGGAGGCCGATCACGGCCACCACGGACTTGAAGCCCTGGGCCTCCATGCTCTTGAGGAGGTGGGTGTAGAGGGTGGAGCCGAGGCCGAGGCGCTGGTGGCGGTGGGACACGTACACGGTGGACTCCACGGTCCAGTCGTAGGCGTTGCGGGCCTTCCACGGGCCGGCGTAGGCGATGCCGGCCACCACGCCCTCCACCTCGGCCACGAGCCACGGGTAGCGGTCCTGGAGGCGCTCCAGGTCGTCGATCCACTCCTGCGGGGTCTGCGGCTCGGTGCGGAAGTTCACGGTGGAGGTCTCGATGTAGTGGTTCACGATGTCGCACACGGCGGCCATGTCGGCGGCGGTGGCCGGGCGGATCTCGACGGGGCGGCGCTCGGGGGACATGGTGTCGTGTGGATCCCGGTGGATCTGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTTCGCTAGCGAATTGATCCTCTAGAGTCGACCTGCAGAAGTAACACCAAACAACAGGGTGAGCATCGACAAAAGAAACAGTACCAAGCAAATAAATAGCGTATGAAGGCAGGGCTAAAAAAATCCACATATAGCTGCTGCATATGCCATCATCCAAGTATATCAAGATCAAAATAATTATAAAACATACTTGTTTATTATAATAGATAGGTACTCAAGGTTAGAGCATATGAATAGATGCTGCATATGCCATCATGTATATGCATCAGTAAAACCCACATCAACATGTATACCTATCCTAGATCGATATTTCCATCCATCTTAAACTCGTAACTATGAAGATGTATGACACACACATACAGTTCCAAAATTAATAAATACACCAGGTAGTTTGAAACAGTATTCTACTCCGATCTAGAACGAATGAACGACCGCCCAACCACACCACATCATCACAACCAAGCGAACAAAAAGCATCTCTGTATATGCATCAGTAAAACCCGCATCAACATGTATACCTATCCTAGATCGATATTTCCATCCATCATCTTCAATTCGTAACTATGAATATGTATGGCACACACATACAGATCCAAAATTAATAAATCCACCAGGTAGTTTGAAACAGAATTCTACTCCGATCTAGAACGACCGCCCAACCAGACCACATCATCACAACCAAGACAAAAAAAAGCATGAAAAGATGACCCGACAAACAAGTGCACGGCATATATTGAAATAAAGGAAAAGGGCAAACCAAACCCTATGCAACGAAACAAAAAAAATCATGAAATCGATCCCGTCTGCGGAACGGCTAGAGCCATCCCAGGATTCCCCAAAGAGAAACACTGGCAAGTTAGCAATCAGAACGTGTCTGACGTACAGGTCGCATCCGTGTACGAACGCTAGCAGCACGGATCTAACACAAACACGGATCTAACACAAACATGAACAGAAGTAGAACTACCGGGCCCTAACCATGCATGGACCGGAACGCCGATCTAGAGAAGGTAGAGAGGGGGGGGGGGGAGGACGAGCGGCGTACCTTGAAGCGGAGGTGCCGACGGGTGGATTTGGGGGAGATCTGGTTGTGTGTGTGTGCGCTCCGAACAACACGAGGTTGGGGAAAGAGGGTGTGGAGGGGGTGTCTATTTATTACGGCGGGCGAGGAAGGGAAAGCGAAGGAGCGGTGGGAAAGGAATCCCCCGTAGCTGCCGGTGCCGTGAGAGGAGGAGGAGGCCGCCTGCCGTGCCGGCTCACGTCTGCCGCTCCGCCACGCAATTTCTGGATGCCGACAGCGGAGCAAGTCCAACGGTGGAGCGGAACTCTCGAGAGGGGTCCAGAGGCAGCGACAGAGATGCCGTGCCGTCTGCTTCGCTTGGCCCGACGCGACGCTGCTGGTTCGCTGGTTGGTGTCCGTTAGACTCGTCGACGGCGTTTAACAGGCTGGCATTATCTACTCGAAACAAGAAAAATGTTTCCTTAGTTTTTTTAATTTCTTAAAGGGTATTTGTTTAATTTTTAGTCACTTTATTTTATTCTATTTTATATCTAAATTATTAAATAAAAAAACTAAAATAGAGTTTTAGTTTTCTTAATTTAGAGGCTAAAATAGAATAAAATAGATGTACTAAAAAAATTAGTCTATAAAAACCATTAACCCTAAACCCTAAATGGATGTACTAATAAAATGGATGAAGTATTATATAGGTGAAGCTATTTGCAAAAAAAAAGGAGAACACATGCACACTAAAAAGATAAAACTGTAGAGTCCTGTTGTCAAAATACTCAATTGTCCTTTAGACCATGTCTAACTGTTCATTTATATGATTCTCTAAAACACTGATATTATTGTAGTACTATAGATTATATTATTCGTAGAGTAAAGTTTAAATATATGTATAAAGATAGATAAACTGCACTTCAAACAAGTGTGACAAAAAAAATATGTGGTAATTTTTTATAACTTAGACATGCAATGCTCATTATCTCTAGAGAGGGGCACGACCGGGTCACGCTGCACTGCAGGCTAGCGGCGAATTCGCCCTTGTACGCGTACGCGTATATATACGTGCGCTGCTACTCATTTGCGCGGGAATACAGCTCAGTCTGCTGTGCGCTGCAGGATGTACATACATACATGCGCAGGTGCAAAGTCTACGCGCGCGGGCAATGCAAGCCCCTGGCGTAGTTGGGCCATGACTGAGATCACGCCTCATGGTCATGGAACGAAACACCGCGTCCGGCCGGGCTGCCCCTGGCGTCACGCGGGAGGCAGCTGCTAGCGTTAGCGTACGTACCCACCGTCTCGTACACACCACCGCAGGGAGAGAGAAGAGCGATGCAATGCACATGTACAGCATCCGCATCATGCATAGATACTCATATCTTCAAGGCCACACATGCAGCAGTGTCGTACGCTACGTTGTTTCAACGGAGGAGGAGGATACATACATAGACACCCACAGCCAGCCTAGCATATAGCAGATAGCATACGGACTCCCGGGTGAGGAAAAATGGAGGGCGAACCAAACCAACCACAAAGAAGCAGCAGCAGCAGCAGCAGCAGCTGCGGCTGCTATCACCACTCACCAACTCCAATTAAAGATCTCTCTCTCTCTCTCTACTGGCCGGCCCTGTCAGTGCCAGCGCCCGGTTTGTTGCTAGCTGAGCTGCGGGCGTCGCTCTTAGATATAGCCCAAAACTCACTCCACCACCACTCGTTCCATGGAACCCTAGACCAAAAGTACTCGCGCTCTCGGCCCTCGCTCTCGCCCTCTCCCTCTCCGCAGCAAAAGAGATCCGGCCGGCCGAGAAGGGCGCGCGCTAGCTGCCCGGCTACTAGCTGGCGCCCGCCCGCGCATATATCTGTGTCATCGCCATCACCCACACCATGGCCCGGCCGGCCAACACCGCCGTATTAGCTCTGTCTGTCGCTCGTCCACCTGCGACCGACTGAGCGATCGATCTCCACCGAGCTCTCCGCTAAGCGCTGTCCTTGCCGCCGTCCTCCCCTCCGTCCCCTACGCATCCATTTCCGTGTGCTCGTGTGTGCGCGCGCGGGCACTCCTGCTCCTGCTCCCTCCGGCCCCTCCTCCCCTCCCAGGCTCCCAGCTAGCCGCGCCCGCCCGCGCGACCTGCACCTGCACAGATCGGGCGGCCGGGCCGACCGATCGATCGAGAT

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

ATGGCACCGAAGAAGAAGCGCAAGGTGATGGACAAGAAGTACAGCATCGGCCTCGACATCGGCACCAACTCGGTGGGCTGGGCCGTCATCACGGACGAATATAAGGTCCCG

TCGAAGAAGTTCAAGGTCCTCGGCAATACAGACCGCCACAGCATCAAGAAAAACTTGATCGGCGCCCTCCTGTTCGATAGCGGCGAGACCGCGGAGGCGACCAGGCTCAAG

AGGACCGCCAGGAGACGGTACACTAGGCGCAAGAACAGGATCTGCTACCTGCAGGAGATCTTCAGCAACGAGATGGCGAAGGTGGACGACTCCTTCTTCCACCGCCTGGAG

GAATCATTCCTGGTGGAGGAGGACAAGAAGCATGAGCGGCACCCAATCTTCGGCAACATCGTCGACGAGGTAAGTTTCTGCTTCTACCTTTGATATATATATAATAATTAT

CATTAATTAGTAGTAATATAATATTTCAAATATTTTTTTCAAAATAAAAGAATGTAGTATATAGCAATTGCTTTTCTGTAGTTTATAAGTGTGTATATTTTAATTTATAAC

TTTTCTAATATATGACCAAAACATGGTGATGTGCAGGTGGCCTACCACGAGAAGTACCCGACAATCTACCACCTCCGGAAGAAACTGGTGGACAGCACAGACAAGGCGGAC

CTCCGGCTCATCTACCTTGCCCTCGCGCATATGATCAAGTTCCGCGGCCACTTCCTCATCGAGGGCGACCTGAACCCGGACAACTCCGACGTGGACAAGCTGTTCATCCAG

CTCGTGCAGACGTACAATCAACTGTTCGAGGAGAACCCCATAAACGCTAGCGGCGTGGACGCCAAGGCCATCCTCTCGGCCAGGCTCTCGAAATCAAGAAGGCTGGAGAAC

CTTATCGCGCAGTTGCCAGGCGAAAAGAAGAACGGCCTCTTCGGCAACCTTATTGCGCTCAGCCTCGGCCTGACGCCGAACTTCAAATCAAACTTCGACCTCGCGGAGGAC

GCCAAGCTCCAGCTCTCAAAGGACACCTACGACGACGACCTCGACAACCTCCTGGCCCAGATAGGAGACCAGTACGCGGACCTCTTCCTCGCCGCCAAGAACCTCTCCGAC

GCTATCCTGCTCAGCGACATCCTTCGGGTCAACACCGAAATTACCAAGGCACCGCTGTCCGCCAGCATGATTAAACGCTACGACGAGCACCATCAGGACCTCACGCTGCTC

AAGGCACTCGTCCGCCAGCAGCTCCCCGAGAAGTACAAGGAGATCTTCTTCGACCAATCAAAAAACGGCTACGCGGGATATATCGACGGCGGTGCCAGCCAGGAAGAGTTC

TACAAGTTCATCAAACCAATCCTGGAGAAGATGGACGGCACCGAGGAGTTGCTGGTCAAGCTCAACAGGGAGGACCTCCTCAGGAAGCAGAGGACCTTCGACAACGGCTCC

ATCCCGCATCAGATCCACCTGGGCGAACTGCATGCCATCCTGCGGCGCCAGGAGGACTTCTACCCGTTCCTGAAGGATAACCGGGAGAAGATCGAGAAGATCTTGACGTTC

CGCATCCCATACTACGTGGGCCCGCTGGCTCGCGGCAACTCCCGGTTCGCCTGGATGACCCGGAAGTCGGAGGAGACCATCACACCCTGGAACTTTGAGGAGGTGGTCGAT

AAGGGCGCTAGCGCTCAGAGCTTCATCGAGCGCATGACCAACTTCGATAAAAACCTGCCCAATGAAAAAGTCCTCCCCAAGCACTCGCTGCTCTACGAGTACTTCACCGTG

TACAACGAGCTCACCAAGGTCAAATACGTCACCGAGGGCATGCGGAAGCCGGCGTTCCTGAGCGGCGAGCAGAAGAAGGCGATAGTGGACCTCCTCTTCAAGACCAACAGG

AAGGTGACCGTGAAGCAATTAAAAGAGGACTACTTCAAGAAAATAGAGTGCTTCGACTCCGTGGAGATCTCGGGCGTGGAGGATCGGTTCAACGCCTCACTCGGCACGTAT

CACGACCTCCTCAAGATCATTAAAGACAAGGACTTCCTCGACAACGAGGAGAACGAGGACATCCTCGAGGACATCGTCCTCACCCTGACCCTGTTCGAGGACCGCGAAATG

ATCGAGGAGAGGCTGAAGACCTACGCGCACCTGTTCGACGACAAGGTCATGAAACAGCTCAAGAGGCGCCGCTACACTGGTTGGGGAAGGCTGTCCCGCAAGCTCATTAAT

GGCATCAGGGACAAGCAGAGCGGCAAGACCATCCTGGACTTCCTCAAGTCCGACGGGTTCGCCAACCGCAACTTCATGCAGCTCATTCACGACGACTCGCTCACGTTCAAG

GAAGACATCCAGAAGGCACAGGTGAGCGGGCAGGGTGACTCCCTCCACGAACACATCGCCAACCTGGCCGGCTCGCCGGCCATTAAAAAGGGCATCCTGCAGACGGTCAAG

GTCGTCGACGAGCTCGTGAAGGTGATGGGCCGGCACAAGCCCGAAAATATCGTCATAGAGATGGCCAGGGAGAACCAGACCACCCAAAAAGGGCAGAAGAACTCGCGCGAG

CGGATGAAACGGATCGAGGAGGGCATTAAAGAGCTCGGGTCCCAGATCCTGAAGGAGCACCCCGTGGAAAATACCCAGCTCCAGAATGAAAAGCTCTACCTCTACTACCTG

CAGAACGGCCGCGACATGTACGTGGACCAGGAGCTGGACATTAATCGGCTATCGGACTACGACGTCGACCACATCGTGCCGCAGTCGTTCCTCAAGGACGATAGCATCGAC

AACAAGGTGCTCACCCGGTCGGATAAAAATCGGGGCAAGAGCGACAACGTGCCCAGCGAGGAGGTCGTGAAGAAGATGAAAAACTACTGGCGCCAGCTCCTCAACGCGAAA

CTGATCACCCAGCGCAAGTTCGACAACCTGACGAAGGCGGAACGCGGTGGCTTGAGCGAACTCGATAAGGCGGGCTTCATAAAAAGGCAGCTGGTCGAGACGCGCCAGATC

ACGAAGCATGTCGCCCAGATCCTGGACAGCCGCATGAATACTAAGTACGATGAAAACGACAAGCTGATCCGGGAGGTGAAGGTGATCACGCTGAAGTCCAAGCTCGTGTCG

GACTTCCGCAAGGACTTCCAGTTCTACAAGGTCCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAATGCGGTGGTCGGGACCGCCCTGATCAAGAAGTAC

CCGAAGCTGGAGTCGGAGTTCGTGTACGGCGACTACAAGGTCTACGACGTGCGCAAAATGATCGCCAAGTCCGAGCAGGAGATCGGCAAGGCCACGGCAAAATACTTCTTC

TACTCGAACATCATGAACTTCTTCAAGACCGAGATCACCCTCGCGAACGGCGAGATCCGCAAGCGCCCGCTCATCGAAACCAACGGCGAGACGGGCGAGATCGTCTGGGAT

AAGGGCCGGGATTTCGCGACGGTCCGCAAGGTGCTCTCCATGCCGCAAGTCAATATCGTGAAAAAGACGGAGGTCCAGACGGGCGGGTTCAGCAAGGAGTCCATCCTCCCG

AAGCGCAACTCCGACAAGCTCATCGCGAGGAAGAAGGATTGGGACCCGAAAAAATATGGCGGCTTCGACAGCCCGACCGTCGCATACAGCGTCCTCGTCGTGGCGAAGGTG

GAGAAGGGCAAGTCAAAGAAGCTCAAGTCCGTGAAGGAGCTGCTCGGGATCACGATTATGGAGCGGTCCTCCTTCGAGAAGAACCCGATCGACTTCCTAGAGGCCAAGGGA

TATAAGGAGGTCAAGAAGGACCTGATTATTAAACTGCCGAAGTACTCGCTCTTCGAGCTGGAAAACGGCCGCAAGAGGATGCTCGCCTCCGCAGGCGAGTTGCAGAAGGGC

AACGAGCTCGCCCTCCCGAGCAAATACGTCAATTTCCTGTACCTCGCTAGCCACTATGAAAAGCTCAAGGGCAGCCCGGAGGACAACGAGCAGAAGCAGCTCTTCGTGGAG

CAGCACAAGCATTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCGAAGCGGGTGATCCTCGCCGACGCGAACCTGGACAAGGTGCTGTCGGCATATAACAAGCAC

CGCGACAAACCAATACGCGAGCAGGCCGAAAATATCATCCACCTCTTCACCCTCACCAACCTCGGCGCTCCGGCAGCCTTCAAGTACTTCGACACCACGATTGACCGGAAG

CGGTACACGAGCACGAAGGAGGTGCTCGATGCGACGCTGATCCACCAGAGCATCACAGGGCTCTATGAAACACGCATCGACCTGAGCCAGCTGGGCGGAGACAAGAGACCA

CGGGACCGCCACGATGGCGAGCTGGGAGGCCGCAAGCGGGCAAGGTAG

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.

TGAGAGTACAATGATGAACCTAGATTAATCAATGCCAAAGTCTGAAAAATGCACCCTCAGTCTATGATCCAGAAAATCAAGATTGCTTGAGGCCCTG

TTCGGTTGTTCCGGATTAGAGCCCCGGATTAATTCCTAGCCGGATTACTTCTCTAATTTATATAGATTTTGATGAGCTGGAATGAATCCTGGCTTAT

TCCGGTACAACCGAACAGGCCCTGAAGGATACCAGTAATCGCTGAGCTAAATTGGCATGCTGTCAGAGTGTCAGTATTGCAGCAAGGTAGTGAGATA

ACCGGCATCATGGTGCCAGTTTGATGGCACCATTAGGGTTAGAGATGGTGGCCATGGGCGCATGTCCTGGCCAACTTTGTATGATATATGGCAGGGT

GAATAGGAAAGTAAAATTGTATTGTAAAAAGGGATTTCTTCTGTTTGTTAGCGCATGTACAAGGAATGCAAGTTTTGAGCGAGGGGGCATCAAAGAT

CTGGCTGTGTTTCCAGCTGTTTTTGTTAGCCCCATCGAATCCTTGACATAATGATCCCGCTTAAATAAGCAACCTCGCTTGTATAGTTCCTTGTGCT

CTAACACACGATGATGATAAGTCGTAAAATAGTGGTGTCCAAAGAATTTCCAGGCCCAGTTGTAAAAGCTAAAATGCTATTCGAATTTCTACTAGCA

GTAAGTCGTGTTTAGAAATTATTTTTTTATATACCTTTTTTCCTTCTATGTACAGTAGGACACAGTGTCAGCGCCGCGTTGACGGAGAATATTTGCA

AAAAAGTAAAAGAGAAAGTCATAGCGGCGTATGTGCCAAAAACTTCGTCACAGAGAGGGCCATAAGAAACATGGCCCACGGCCCAATACGAAGCACC

GCGACGAAGCCCAAACAGCAGTCCGTAGGTGGAGCAAAGCGCTGGGTAATACGCAAACGTTTTGTCCCACCTTGACTAATCACAAGAGTGGAGCGTA

CCTTATAAACCGAGCCGCAAGCACCGAATTGCGTACGCGTACGTGTGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAAC

TTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTTT