CBI-DELETED COPY

June 12, 2017

Dr. Michael J. Firko APHIS Deputy Administrator Regulatory Services 4700 River Rd, Unit 98 Riverdale, MD 20737

Re: Confirmation of Regulatory Status of a Genome-Edited 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 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 in the family that is not on the USDA federal noxious 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 [ CBI-deleted ]. The inactivation of the [ ) gene is thus expected to CBI-deleted ]. It has been shown previously by other researchers that the Cas9 CBI-deleted 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 CBI-deleted nucleotide insertion that disrupts the coding sequence of the gene. As described below, line [ ) 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 [ CBI-deleted ] via inactivation of the endogenous [ ] gene that CBI-deleted contributes to [ ]. Because camelina is an allohexaploid, there are CBI-deleted

Yield10 Bioscience, Inc. 19 Presidential Way, Suite 201 Woburn, MA 01801 (617) 583-1700 www.yield1obio.com CBI-DELETED COPY

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. CBI-deleted

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 [ CBI-deleted 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 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 [ ], one guide RNA was sufficient to generate CBI-deleted the modification in all six alleles of the [ ] gene. A detailed list of genetic elements, CBI-deleted their origin, and their function is presented in Table l. In short, the T-DNA of binary vector [ ] carries three expression cassettes: CBI-deleted

- An expression cassette for the two exons of Cas9 endonuclease from Streptococcus pyogenes [ CBI-deleted ]. The expression of the Cas9 gene is controlled by the [ CBI-deleted

- 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

2 CBI-DELETED COPY

Table 1. Genetic Elements of [ ] used to create line [ ] . The vector backbone 2x CBI-deleted (region outside of the T-DNA) is identical to standard binary vector [ CBI-deleted 1.

Genetic Source Function Element U6-26 [ Polymerase Ill promoter of the U6-26 small nuclear CBI-deleted promoter J RNA gene to drive t ranscription of cassette 1 composed of the [ I #4 spacer and the guide CBI-deleted RNA scaffold which together encode the functional chimeric guide RNA [ I Camelina sativa Encodes [ 2x CBI-deleted .... #4 spacer QI ... ]. This "spacer" directs QI the Cas9 endonuclease to the [ genes for CBI-deleted "'ro I u cleavage Guide RNA Streptococcus Encodes crRNA-tracrRNA fusion transcript that is scaffold pyogenes necessary for Cas9 binding. The [ I #4 spacer CBI-deleted and the guide RNA scaffold together constitute a functional chimeric guide RNA U6-26 [ Terminator of U6-26 small nuclear RNA polymerase Ill CBI-deleted J to terminate transcription of [ I #4 spacer CBI-deleted and guide RNA scaffold [ I [ [ 3x CBI-deleted promoter

J ], controls expression of the Cas9 coding sequence. [ l [ [ I 3x CBI-deleted I [ [ I [ 3x CBI-deleted N ...QI I I OJ "' [ I Streptococcus Exon 1 of Cas9 endonuclease [ 2x CBI-deleted "'ro u pyogenes I [ I Solanum [ 2x CBI-deleted tuberosum I to optimize Cas9 expression in [ I Streptococcus Exon 2 of Cas9 endonuclease [ 2x CBI-deleted pyogenes I [ [ [ I J 3x CBI-deleted

l [ I [ Terminator of the [ l to 3x CBI-deleted terminator J terminate transcription of Cas9

3 CBI-DELETED COPY

I I I I I promoter, drives expression of the 3x CBI-deleted promoter I I I selection marker CBI-deleted I [ J [ 3x CBI-deleted

!Y') I ...Q) selection Q) V, marker V, uC'tl

I [ I I Terminator of the [ I gene to terminate 3x CBI-deleted terminator I transcription of the [ ) selection marker CBI-deleted 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 tra nsfer of the Border tumefaciens Ti T-DNA into the plant cell genome plasmid

The binary vector backbone is identical to the standard binary vector [ CBI-deleted ] and includes both T-DNA borders.

The next step comprised [ CBI-deleted ], indicating the presence of the T-DNA containing the Cas9 expression and guide RNA expression cassettes. Tl seeds were planted in soil and tissue was screened for genome editing by Amplicon 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 Iicon 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

4 CBI-DELETED COPY

the three loci suggesting that line [ ] is completely edited within detection limits of the CBI-deleted Amplicon sequencing.

(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:

5 CBI-DELETED COPY

Reference Date Species Firko to Epicrop technologies April 7, 2017 Glycine max - Firko to Iowa State University May 22, 2015 Oryza sativa - Gregoire to University of Nebraska June 6, 2012

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 [ ] does not contain any foreign CBI-deleted DNA or plant pest sequences.

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 CBI-deleted Part 340.

We look forward to answering any questions you might have.

Sincerely,

Karen Bohmert-Tatarev, PhD Kristi D. Snell, PhD Technology Manager VP of Research & CSO 617-583-1769 617-583-1729 [email protected] [email protected]

References:

CBI-deleted

6 CBI-DELETED COPY

7