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
7
Embed
CBI-DELETED COPY...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
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
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
caeck
Received
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