Optimization of T-DNA architecture for Cas9-mediated mutagenesis in Arabidopsis

Abstract

Bacterial CRISPR systems have been widely adopted to create operator-specified site-specific nucleases. Such nuclease action commonly results in loss-of-function alleles, facilitating functional analysis of genes and gene families We conducted a systematic comparison of components and T-DNA architectures for CRISPR-mediated gene editing in Arabidopsis, testing multiple promoters, terminators, sgRNA backbones and Cas9 alleles. We identified a T-DNA architecture that usually results in stable (i.e. homozygous) mutations in the first generation after transformation. Notably, the transcription of sgRNA and Cas9 in head-to-head divergent orientation usually resulted in highly active lines. Our Arabidopsis data may prove useful for optimization of CRISPR methods in other plants.

Fig 1. Golden Gate cloning method enables assembly of CRISPR modules in various combinations.

Cas9 alleles, promoters and terminators were cloned into the indicated Level 0 acceptor vectors as described in Materials and Methods and were assembled in Level 1 acceptor vector pICH47742. sgRNAs targeting AtADH1 were amplified by PCR and assembled with the U6-26 promoter vector pICSL90002 in the same manner. Both Cas9 and sgRNA expression units were assembled in Level 2 acceptors pAGM4723 (not containing an overdrive sequence) or pICSL4723 (containing an overdrive) along with a Glufosinate resistance plant selectable marker. An end-linker pICH41766 (EL2;3) was used to link the sgRNA expression unit to the Level 2 acceptor vector. For a “head-to-head orientation” of the sgRNA and Cas9 expression cassettes, Cas9 allele, promoter and terminator were assembled into pICH47811 instead of pICH47742.

Fig 2. Evaluation of mutation rates.

Constructs were transformed into Arabidopsis accession Col-0 via Agrobacterium tumefaciens strain GV3101. Six independent transformants (T1) were selected using Glufosinate. About 100 progeny (T2) of each transformant were selected for allyl-alcohol resistance. For each independent T2 family, up to six allyl-alcohol resistant plants were genotyped at the ADH1 locus. For each T2 family, the mutation rate was calculated as [(number of allyl-alcohol surviving plants) x (% of homozygous or biallelic mutants confirmed by sequencing among the surviving plants tested) / (number of seeds sown)].

Fig 3. UBI10, YAO and RPS5a promoter-regulated Cas9 expression enhances mutation rates.

a. to h. Each panel represents a promoter comparison in the same T-DNA context. Promoters can be compared within each panel, but not from one panel to another. The modules were assembled into pICSL4723 (RB+OD, with an overdrive) or pAGM4723 (RB, without an overdrive) and transformed into Col-0 via Agrobacterium tumefaciens strain GV3101. LB: Left Border. SM: Selectable Marker (Glufosinate resistance gene). 35S: 426 bp of the 35S promoter from Cauliflower Mosaic Virus. UBI10: 1327 bp of the At4g05320 promoter. EC1.2: 1014 bp of the At2g21740 promoter. EC_enh.: 752 bp of the At2g21740 promoter fused to 548 bp of the At1g76750 promoter. MGE1: 1554 bp of the At5g55200 promoter. AG: 3101 bp of the At4g18960 promoter. ICU2: 625 bp of the At5g67100 promoter. CsVMV: 517 bp of a promoter from Cassava Vein Mosaic virus. RPS5a: 1688 bp of the At3g11940 promoter. YAO: 596 bp of the At4g05410 promoter. Cas9_1: Mali et al., 2013 [3]. Cas9_2: Fauser et al., 2014 [13]. Cas9_3: Li et al., 2013 [25]. Cas9_4: Le Cong et al., 2013 [10]. E9T: 631 bp of the Pisum sativum rbcS E9 terminator. OcsT: 714 bp of the Agrobacterium tumefaciens octopine synthase terminator. AgsT: 410 bp of the Agrobacterium tumefaciens agropine synthase terminator. NosT: 267 bp of the Agrobacterium tumefaciens nopaline synthase terminator. pU6-26: 205 bp of the At3g13855 promoter. sgRNA^EF: “extension-flip” sgRNA. U6-26T: 7, 67 or 192 bp of the At3g13855 terminator. RB: Right Border. d. Five lines were tested for UBI10 and ICU2 and four lines for AG instead of six. F. Five lines were tested for YAO and RPS5a instead of six. The sgRNA targets ADH1. CRISPR activity measured in % of homozygous or biallelic stable mutants in the second generation after transformation (T2). Each dot represents an independent T2 family. Red dot: All the T2 lines from this family carry the same mutation, indicating a mutation more likely inherited from the T1 parent rather than being de novo from the T2 line. Bold and underlined: Most active construct(s) for each panel.

Fig 4. An intron-containing allele of Cas9 triggers elevated mutation rates.

a. Cas9_1: Mali et al., 2013 [3]. Cas9_2: Fauser et al., 2014 [13]. Cas9_3: Li et al., 2013 [25]. Cas9_4: Le Cong et al., 2013 [10]. NLS: Nuclear Localization Signal. FLAG: DYKDDDDK peptide. Apart from the FLAG and NLS, the amino acid sequences are identical. The nucleotide sequence (codon optimization) are different. Bars are not in scale. b. to h. Each panel represents a CDS comparison in the same context. CDSs can be compared within each panel, not from one panel to another. The modules were assembled into pICSL4723 (RB+OD, with an overdrive) or pAGM4723 (RB, without an overdrive) and transformed into Col-0 via Agrobacterium tumefaciens strain GV3101. LB: Left Border. SM: Selectable Marker (Glufosinate resistance gene). pEC_enh.: 752 bp of the At2g21740 promoter fused to 548 bp of the At1g76750 promoter. pYAO: 596 bp of the At4g05410 promoter. pRPS5a: 1688 bp of the At3g11940 promoter. CsVMV: 517 bp of a promoter from Cassava Vein Mosaic virus. pICU2: 625 bp of the At5g67100 promoter. E9T: 631 bp of the Pisum sativum rbcS E9 terminator. OcsT: 714 bp of the Agrobacterium tumefaciens octopine synthase terminator. U6-26p: 205 bp of the At3g13855 promoter. sgRNA^EF: “extension-flip” sgRNA. U6-26T: 7, 67 or 192 bp of the At3g13855 terminator. RB: Right Border. b. Cas9_2 is in pAGM4723 (i.e. RB) in combination with U6-26¹⁹²; Cas9_3 and Cas9_4 are in pICSL4723 (i.e. RB+OD) in combination with U6-26⁶⁷. c. and d. Five lines were tested for Cas9_3 instead of six. The sgRNA targets ADH1. CRISPR activity measured in % of homozygous or biallelic stable mutants in the second generation after transformation (T2). Each dot represents an independent T2 family. Red dot: All the T2 lines from this family carry the same mutation, indicating a mutation more likely inherited from the T1 parent rather than being de novo from the T2 line. Bold and underlined: Most active construct(s) for each panel.

Fig 5. A modified sgRNA is slightly more efficient to trigger mutations.

a. Original sgRNA proposed by Mali et al., 2013 [3]. Extension-Flip (EF) sgRNA proposed by Chen et al., 2013 [8]. b. and c. Each panel represents a sgRNA backbone comparison in the same context. sgRNA backbones can be compared within each panel but not from one panel to another. The modules were assembled into pICSL4723 (RB+OD, with an overdrive) or pAGM4723 (RB, without an overdrive) and transformed into Col-0 via Agrobacterium tumefaciens strain GV3101. LB: Left Border. SM: Selectable Marker (Glufosinate resistance gene). CsVMV: 517 bp of a promoter from Cassava Vein Mosaic virus. UBI10: 1327 bp of the At4g05320 promoter. Cas9_2: Fauser et al., 2014 [13]. OcsT: 714 bp of the Agrobacterium tumefaciens octopine synthase terminator. U6-26p: 205 bp of the At3g13855 promoter. U6-26T: 7 bp of the At3g13855 terminator. RB: Right Border. c. Five lines were tested for sgRNA^EF and four lines for sgRNA instead of six. The sgRNA targets ADH1. CRISPR activity measured in % of homozygous or biallelic stable mutants in the second generation after transformation (T2). Each dot represents an independent T2 family. Bold and underlined: Most active construct(s) for each panel.

Fig 6. The sgRNA expression regulated by an authentic 3’ regulatory sequence of U6-26 produces greater mutation rates.

a. to c. Each panel represents a terminator comparison in the same context. Terminators can be compared within each panel, not from one panel to another. The modules were assembled into pAGM4723 and transformed into Col-0 via Agrobacterium tumefaciens strain GV3101. LB: Left Border. SM: Selectable Marker (Glufosinate resistance gene). ICU2: 625 bp of the At5g67100 promoter. 35S: 426 bp of the 35S promoter from Cauliflower Mosaic Virus. CsVMV: 517 bp of a promoter from Cassava Vein Mosaic virus. Cas9_2: Fauser et al., 2014 [13]. Cas9_3: Li et al., 2013 [25]. OcsT: 714 bp of the Agrobacterium tumefaciens octopine synthase terminator. AgsT: 410 bp of the Agrobacterium tumefaciens agropine synthase terminator. U6-26p: 205 bp of the At3g13855 promoter. sgRNA^EF: “extension-flip” sgRNA. U6-26T: 7 or 192 bp of the At3g13855 terminator. RB: Right Border. a. Five lines were tested for U6-26⁷ instead of six. The sgRNA targets ADH1. CRISPR activity measured in % of homozygous or biallelic stable mutants in the second generation after transformation (T2). Each dot represents an independent T2 family. Bold and underlined: Most active construct(s) for each panel.

Fig 7. A weak 3’ regulatory sequence reduces the CRISPR-induced mutation rate.

a. to c. Each panel represents a terminator comparison in the same context. Terminators can be compared within each panel, not from one panel to another. The modules were assembled into pAGM4723 and transformed into Col-0 via Agrobacterium tumefaciens strain GV3101. LB: Left Border. SM: Selectable Marker (Glufosinate resistance gene). EC_enh.: 752 bp of the At2g21740 promoter fused to 548 bp of the At1g76750 promoter. UBI10: 1327 bp of the At4g05320 promoter. Cas9_2: Fauser et al., 2014 [13]. Cas9_3: Li et al., 2013 [25]. E9T: 631 bp of the Pisum sativum rbcS E9 terminator. OcsT: 714 bp of the Agrobacterium tumefaciens octopine synthase terminator. AgsT: 410 bp of the Agrobacterium tumefaciens agropine synthase terminator. U6-26p: 205 bp of the At3g13855 promoter. sgRNA^EF: “extension-flip” sgRNA. U6-26T: 7, 67 or 192 bp of the At3g13855 terminator. RB: Right Border. For the comparison using the UBI10 promoter, the AgsT is in combination with U6-26¹⁹²; OcsT is in combination with U6-26⁶⁷. The sgRNA targets ADH1. CRISPR activity measured in % of homozygous or biallelic stable mutants in the second generation after transformation (T2). Each dot represents an independent T2 family. Red dot: All the T2 lines from this family carry the same mutation, indicating a mutation more likely inherited from the T1 parent rather than being de novo from the T2 line. Bold and underlined: Most active construct(s) for each panel.

Fig 8. CRISPR activity is similar or higher when the sgRNA and the Cas9 expression cassettes are in a head-to-head orientation.

a. and b. Each panel represents an orientation comparison in the same context. Orientations can be compared within each panel, not from one panel to another. The modules have been assembled by Golden Gate into pICSL4723 (RB+OD, with an overdrive) and transformed into Col-0 via Agrobacterium tumefaciens strain GV3101. LB: Left Border. SM: Selectable Marker (Glufosinate resistance gene. RPS5a: 1688 bp of the At3g11940 promoter. YAO: 596 bp of the At4g05410 promoter. Cas9_3: Li et al., 2013 [25]. Cas9_4: Le Cong et al., 2013 [10]. E9T: 631 bp of the Pisum sativum rbcS E9 terminator. U6-26p: 205 bp of the At3g13855 promoter. sgRNA^EF: “extension-flip” sgRNA. U6-26T: 67 bp of the At3g13855 terminator. RB: Right Border. a. Five lines were tested for H2H instead of six. b. Five lines were tested for H2T instead of six. The sgRNA targets ADH1. CRISPR activity measured in % of homozygous or biallelic stable mutants in the second generation after transformation (T2). Each dot represents an independent T2 family. Red dot: All the T2 lines from this family carry the same mutation, indicating a mutation more likely inherited from the T1 parent rather than being de novo from the T2 line. Bold and underlined: Most active construct(s) for each panel.

Fig 9. Genotype at ADH1 locus confirmed by capillary sequencing.

For each T2 family tested, up to six allyl-alcohol resistant plants were genotyped by capillary sequencing of an sgRNA target (ADH1) PCR amplicon. We retrieved a total of 315 sequences with a mutation. 59% (187) showed a single sequencing signal, different than ADH1 WT and were classified as “Homozygous”. 11% (33) showed an overlap of two sequencing signals, one matching ADH1 WT and one different; and were classified as “Heterozygous”. 10% (31) showed an overlap of two sequencing signals, none matching ADH1 WT; and were classified as “Biallelic”. 64 (20%) showed an overlap of signals different than WT but not clear enough to distinguish; and were classified as “Unknown”. The “Unknown” sequences can be biallelic or due to somatic mutations but are different than WT, heterozygous or homozygous genotypes.

Fig 10. FAST-Red combined with CRISPR to generate T-DNA free mutants.

The five T1 lines are independent transformants. They are all hemizygous for the T-DNA. At the sgRNA target site, they can be WT, or display somatic, heterozygous, biallelic of homozygous mutations. All the possibilities are represented here. “Somatic” describes events happening in somatic cells, not inherited in the next generation. As somatic events can happen independently in each cell, they often result in mosaic pattern of mutations across the leaf. One line has homozygous mutation (mut1/mut1). It produces seeds segregating for the T-DNA, visible under microscope if using FAST-Red. The seeds will segregate 3:1 (Red: Non-red) if there is one locus insertion, 15:1 (Red: Non-red) if there are two loci insertion, etc. The T2 progeny of (mut1/mut1) is 100% homozygous for the mutation. The non-red seeds are also T-DNA free.