METHOD FOR NUCLEAR GENOME EDITING USING PLASTID SELECTABLE MARKERS
The invention provides methods of using chloroplast-selectable markers to detect nuclear genome editing. The methods involve selecting a genetic modification that has occurred in a nuclear genome of a photosynthetic cell; transforming a photosynthetic cell with a ribonucleoprotein complex that effects a genetic modification in the genome in the nucleus of a photosynthetic cell; transforming the photosynthetic cell with a construct that effects an insertion of a selectable marker into the plastome of a plastid of the cell; selecting for photosynthetic cells having the selectable marker inserted into the plastome; and thereby selecting for the genetic modification to the genome in the nucleus of the photosynthetic cell.
1 . A method of selecting for a genetic modification to a genome in the nucleus of a photosynthetic cell comprising:
transforming the photosynthetic cell with a ribonucleoprotein complex that effects a genetic modification in the genome in the nucleus of the photosoynthetic cell;
transforming the photosynthetic cell with a construct that effects an insertion of a selectable marker into the plastid of the photosynthetic cell;
selecting for photosynthetic cells comprising the selectable marker inserted into the plastid;
thereby selecting for the genetic modification to the genome in the nucleus of the photosynthetic cell.
2 . The method of claim 1 , wherein the insertion of the selectable marker into the plastid of the cell comprises inserting the selectable marker into the plastome.
3 . The method of claim 1 , wherein the plastid is a chloroplast.
4 . The method of claim 1 , further comprising sequencing at least a portion of the genome in the nucleus of the photosynthetic cell to confirm the genetic modification has been performed.
5 . The method of claim 1 , wherein selecting for photosynthetic cells comprises plating algal cells and selecting colonies comprising the insertion into the plastome.
6 . The method of claim 1 , wherein the selectable marker inserted into the plastome enables the photosynthetic cell to grow on an antibiotic.
7 . The method of claim 1 , wherein the antibiotic is erythromycin or spectinomycin.
8 . The method of claim 1 , wherein the selectable marker is glufosinate ammonium or the ptxD gene.
9 . The method of claim 1 , wherein the cells are plated onto selection agar plates.
10 . The method of claim 1 , wherein the ribonucleoprotein complex comprises a guide RNA targeted to a sequence on the genome in the nucleus of the photosynthetic cell and a Cas nuclease.
11 . The method of claim 1 , wherein the method does not comprise screening for a selectable marker comprised in the genome of the nucleus of the photosynthetic cell.
12 . The method of claim 1 , wherein the photosynthetic cell is an algal cell, and the algal cell is a Trebouxiophyte algal organism.
13 . The method of claim 12 , wherein the Trebouxiophyte algal organism is from the genus Oocystis.
14 . The method of claim 1 , wherein the construct that effects an insertion of a selectable marker into the plastid comprises RS-up and RS-down arms that recombine with homologous sequences in the plastome to thereby effect insertion; and the construct further comprises a selectable marker cassette in between the RS-up and RS-down arms.
15 . The method of claim 1 , any one of claims 1-14 wherein the construct is amplified by PCR prior to being transformed into the plastid.
16 . The method of claim 1 , wherein the construct further comprises a chloroplast promoter and chloroplast terminator that controls expression of the encoded selectable marker.
17 . The method of claim 16 , wherein the encoded selectable marker confers the ability to grow on media containing erythromycin, spectinomycin, or glufosinate ammonium.
18 . The method of claim 16 wherein the encoded selectable marker comprises the ptxD gene.
19 . The method of claim 1 , wherein the transformations are performed using biolistics.
20 . A composition comprising a ribonucleoprotein complex for editing a nuclear genome, and a construct comprising a selectable marker gene comprised in between two restriction site arms, each restriction site arm being homologous to corresponding sequences on a plastid genome.
21 . The composition of claim 20 , wherein the ribonucleoprotein complex comprises a guide RNA targeted to a site on the nuclear genome of a subject organism.
22 . The composition of claim 20 , wherein the ribonucleoprotein complex comprises Cas9.
23 . The composition of claim 21 , wherein the subject organism is a Chlorophyte organism.
24 . The composition of claim 23 wherein the Chlorophyte organism is a Trebouxiophyte alga.
25 . The composition of claim 20 , adsorbed to a microcarrier for biolistics.
26 . A kit comprising the composition claim 20 .