June 12, 2017

Dr. Michael J. Firko

APHIS Deputy Administrator

Biotechnology Regulatory Services

4700 River Rd, Unit 98

Riverdale, MD 20737

CBI-DELETED COPY

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

CBI-deleted

CBI-deleted

CBI-deleted

CBI-deleted

[ ) 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

CBI-deleted

CBI-deleted

CBI-deleted

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.

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

CBI-deleted

CBI-deleted

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 [

CBI-deleted

CBI-deleted

CBI-deleted

CBI-deleted

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 [

(region outside of the T-DNA) is identical to standard binary vector [

1.

] . The vector backbone 2x CBI-deleted

CBI-deleted

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

3

CBI-deleted

CBI-deleted

2x CBI-deleted

CBI-deleted

CBI-deleted

CBI-deleted

CBI-deleted

3x CBI-deleted

3x CBI-deleted

3x CBI-deleted

2x CBI-deleted

2x CBI-deleted

2x CBI-deleted

3x CBI-deleted

3x CBI-deleted

!Y')

Q) ... ... Q) V, V, C'tl u

CBI-DELETED COPY

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

3x CBI-deleted CBI-deleted

3x CBI-deleted

3x CBI-deleted

CBI-deleted

CBI-deleted

CBI-deleted

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

4

the three loci suggesting that line [

Amplicon sequencing.

CBI-DELETED COPY

] 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:

5

CBI-DELETED COPY

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

kbohmert-tatarev@yieldlObio.com

References:

6

Kristi D. Snell, PhD

VP of Research & CSO

617-583-1729

snell@yieldlObio.com

CBI-deleted

CBI-deleted

CBI-deleted

CBI-DELETED COPY

7