June 12, 2017
Dr. Michael J. Firko
APHIS Deputy Administrator
Biotechnology Regulatory Services
4700 River Rd, Unit 98
Riverdale, MD 20737
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Re: Confirmation of Regulatory Status of a Genome-Edited Camelina Null Segregant Line
Developed by CRISPR/Cas Technology
Dear Dr. Firko,
YieldlO Bioscience respectfully requests confirmation from USDA-APHIS's Biotechnology
Regulatory Services (BRS) that our genome-edited Camelina sativa (L.) Crantz plant line
developed using the CRISPR/Cas9 genome editing technology does not meet the definition of a
regulated article under 7 CFR Part 340 since the final line does not contain any DNA from a
"plant pest". Camelina sativa is an oil seed crop in the family Brassicaceae that is not on the
USDA federal noxious weed list. The line, herein referred to as line [ ], was developed CBI-deleted
at Metabolix Oilseeds, Inc. {110 Gymnasium Place, Saskatoon, SK, S7N 0W9, Canada), a wholly
owned Canadian subsidiary of Yieldl0 Bioscience. We used disarmed Agrobacterium tumefaciens to deliver a gene encoding the endonuclease Cas9, as well as a cassette coding for a
guide RNA to direct the Cas9 enzyme to a defined site in the plant genome, into plant cells. The
targeted site for genome editing was the [
]. The inactivation of the [ ) gene is thus expected to
]. It has been shown previously by other researchers that the Cas9
enzyme produces double strand DNA breaks that when repaired, incorporate small deletions or
insertions of DNA. DNA sequence analysis has shown that line [ ) contains a single
nucleotide insertion that disrupts the coding sequence of the gene. As described below, line
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[ ) is a null segregant that was obtained using conventional breeding procedures to CBI-deleted
remove the genetic sequences that allow the CRISPR/Cas9 editing to take place such that only
the genome edit, a single nucleotide insertion, remains. Analysis of the parent seed of the line
shows that it produces [ ]. CBI-deleted
Since line [ ) is a null segregant that does not contain any plant pest sequences, and the CBI-deleted
single nucleotide insertion does not generate a plant pest or pose increased weediness
potential, it is our opinion that line [ ) does not meet the definition of a regulated CBI-deleted
article based on 7 CFR Part 340. We however seek confirmation of line [ j's regulatory CBI-deleted status from USDA-APHIS BRS.
Intended Phenotype
The intended phenotype of lines produced from editing of the [
] via inactivation of the endogenous [ ] gene that contributes to [ ]. Because camelina is an allohexaploid, there are
Yield10 Bioscience, Inc. 19 Presidential Way, Suite 201 Woburn, MA 01801 (617) 583-1700
www.yield1obio.com
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three loci for each gene. We generated a line with a single base insertion in all three loci of the
[ ] gene, or six alleles in total.
Intended Activity
Upon confirmation from USDA-APHIS BRS that the edited camelina line is not regulated, YieldlO
Bioscience intends to import the line from its subsidiary in Saskatoon, Canada to its labs in
Woburn, MA. Subsequently we plan to conduct interstate movement and field releases of the line.
Genetic Change in the Final Product
The genetic change is a single base pair insertion in each of the three loci of the [
gene, comprising six alleles in total. These are indistinguishable from changes that could result
from native genome variability, conventional breeding, or chemical or radiation-based mutagenesis.
Development of the Edited Camelina Line
The components for the CRISPR/Cas9 genome editing technology, namely an expression
cassette for the gene encoding the Cas9 endonuclease and an expression cassette for the guide
RNA to target the Cas9 to the desired site in the camelina genome, were delivered into the plant
cells by Agrobacterium-mediated transformation using a binary vector and the floral dip
method. CRISPR/Cas9 is a bacterial endonuclease. It utilizes a combination of protein-DNA and
RNA-DNA pairing to direct targeted double strand breaks in the DNA sequence of interest. The
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guide RNA targets Cas9 to the intended site of action. Due to the design of the [ ] #4 CBI-deleted
spacer used in the development of line [
the modification in all six alleles of the [ ], one guide RNA was sufficient to generate
] gene. A detailed list of genetic elements, their origin, and their function is presented in Table l. In short, the T-DNA of binary vector
[ ] carries three expression cassettes:
- An expression cassette for the two exons of Cas9 endonuclease from Streptococcus
pyogenes [
]. The expression of the Cas9 gene is controlled by the [
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- A cassette encoding the guide RNA [ ] #4 spacer under control of the polymerase Ill CBI-deleted
promoter of the U6-26 small nuclear RNA gene from [ ] CBI-deleted
- An expression cassette for the [ ] selection marker CBI-deleted
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Table 1. Genetic Elements of [ ] used to create line [
(region outside of the T-DNA) is identical to standard binary vector [
1.
] . The vector backbone 2x CBI-deleted
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Genetic Source
Element
U6-26 [ promoter J
[ I Camelina sativa .... #4 spacer QI ... ... QI
"' "' ro u
Guide RNA Streptococcus scaffold pyogenes
U6-26 [
J
[ I [ promoter
J
[ l [
I [ I [
N QI I ... OJ [ I Streptococcus "' "' ro pyogenes u
[ I Solanum tuberosum
[ I Streptococcus pyogenes
[ I [ J
[ I [ terminator J
Function
Polymerase Ill promoter of the U6-26 small nuclear RNA gene to drive t ranscription of cassette 1 composed of the [ I #4 spacer and the guide RNA scaffold which together encode the functional chimeric guide RNA
Encodes [
]. This "spacer" directs the Cas9 endonuclease to the [ I genes for cleavage
Encodes crRNA-tracrRNA fusion transcript that is necessary for Cas9 binding. The [ I #4 spacer and the guide RNA scaffold together constitute a functional chimeric guide RNA
Terminator of U6-26 small nuclear RNA polymerase Ill to terminate transcription of [ I #4 spacer and guide RNA scaffold
[
], controls expression of the Cas9 coding sequence.
[ I
[
I Exon 1 of Cas9 endonuclease [
I [
I to optimize Cas9 expression in plants Exon 2 of Cas9 endonuclease [
I [
l Terminator of the [ l to terminate transcription of Cas9
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3x CBI-deleted
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2x CBI-deleted
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!Y')
Q) ... ... Q) V, V, C'tl u
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I I I I I promoter, drives expression of the promoter I I I selection marker
I [ J [
I selection marker
I [ I I Terminator of the [ I gene to terminate terminator I transcription of the [ ) selection marker
T-DNA Left Agrobacterium Left border of the T-DNA, required for transfer of the Border tumefaciens Ti T-DNA into the plant cell genome
plasmid aphAIII Escherichia coli Bacterial kanamycin resistance marker, provides
K-12 kanamycin resistance for plasmid maintenance in E. coli
pBR322 ori Escherichia coli Bacterial origin of replication from plasmid pMBl, used K-12 for plasmid maintenance in £. coli
pVSrep Pseudomonas Replication protein from plasmid pVSl, used for aeruginosa pVSl plasmid replication in Agrobacterium
pVS sta Pseudomonas Stability protein from plasmid pVSl, used for plasmid aeruginosa pVSl stability in Agrobacterium
T-DNA Right Agrobacterium Right border of the T-DNA, required for t ransfer of the Border tumefaciens Ti T-DNA into the plant cell genome
plasmid
The binary vector backbone is identical to the standard binary vector [
] and includes both T-DNA borders.
The next step comprised [
], indicating the presence of the T-DNA containing the Cas9 expression and guide RNA expression cassettes. Tl
seeds were planted in soil and leaf tissue was screened for genome editing by Amplicon
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sequencing. Plants containing genome edits in the [ ] loci were isolated. Tl plants CBI-deleted
were grown and T2 seed was isolated and screened for [ CBI-deleted
]. The [ ] T2 seeds were CBI-deleted
advanced to generate T3 seeds [ ]. The CBI-deleted
[ ] T3 seeds were advanced to obtain T3 plants. Additional analysis of the null segregant CBI-deleted
plant lines included:
(a) Amp I icon sequencing of all three [ ] loci to determine the nature of the editing in CBI-deleted
all six alleles. Line [ ) carries a single nucleotide insertion in each of its six alleles. No CBI-deleted
wild-type [ ] sequences without the single nucleotide insertion were detected in any of CBI-deleted
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the three loci suggesting that line [
Amplicon sequencing.
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] is completely edited within detection limits of the CBI-deleted
(b) Detailed PCR analysis using 16 primer pairs targeting multiple regions of the T-DNA insert
and the vector backbone. No evidence of any T-DNA or plant vector backbone was detected.
Former USDA-APHIS Jurisdiction on CRISPR/Cas9 Genome Editing
In its responses to previous letters (listed below) USDA-APHIS has concluded that there is no
reason to believe that targeted mutations generated by the gene editing process of Cas9
endonucleases would generate plant pests.
Reference Date Species
Firko to Danforth Center April 7, Setaria viridis 2017
Firko to DuPont Pioneer April 18, Zea mays - corn 2016
Firko to Penn State April 13, Agaricus bisporus - white button University 2016 mushroom
No Plant Pest Sequences Remain in the Edited Camelina Line
The edited camelina line was generated through expression of the Cas9 cassette. The [ CBI-deleted
], pVSl, T-DNA borders and [ ] terminator are derived from plant CBI-deleted
pest sequences ([ ] Pseudomonas aeruginosa, Agrobacterium CBI-deleted
tumefaciens) (Table 1) as designated in 7 CFR 340.2. However, these sequences are not involved
in pathogenicity and do not express proteins that would result in infection or pathogenicity of
the edited camelina line. Importantly, the final edited camelina line, line [ ], is a null CBI-deleted
segregant and does not contain these plant pest sequences but retains the desired single base pair edit.
USDA-APHIS has previously made the determination that genetically modified plants
transformed with Cas9 via Agrobacterium-mediated transformation are not regulated articles if
it was experimentally shown that the Cas9-bearing T-DNA was segregated away from the
targeted mutation through conventional breeding and produced progeny without the inserted
DNA (see letter Firko to Danforth Center, April 7, 2017 on Setaria viridis) .
In addition, USDA-APHIS has previously determined that other null segregants originally
generated via Agrobacterium-mediated transformation are not regulated articles:
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Reference Date Species
Firko to Epicrop technologies April 7, 2017 Glycine max - soybean
Firko to Iowa State University May 22, 2015 Oryza sativa - rice
Gregoire to University of Nebraska June 6, 2012 Sorghum bicolor
Conclusions
Metabolix Oilseeds, a wholly owned Canadian subsidiary of Yieldl0 Bioscience, generated a
CRISPR/Cas9 edited line of the allohexaploid species Camelina sativa. The line, line [ ], CBI-deleted
contains a single base pair insertion in all three loci of the [ ] gene. Line [ is a 2x CBI-deleted
null segregant (genes enabling CRISPR/Cas9 editing were removed through conventional
breeding) yet retains the desired genome edit. Line [
DNA or plant pest sequences. ] does not contain any foreign
In order to facilitate further testing at our Woburn, MA facilities and in the field, YieldlO
Biosciences requests confirmation from Biotechnology Regulatory Services that our edited
camelina line, line [ ], does not meet the definition of a regulated article under 7 CFR Part 340.
We look forward to answering any questions you might have.
Sincerely,
Karen Bohmert-Tatarev, PhD
Technology Manager
617-583-1769
References:
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Kristi D. Snell, PhD
VP of Research & CSO
617-583-1729
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