Vector construction for CRISPR-Cas

mutagenesis in poplar

We modified two plant expression vectors provided by Dr.

Jian‐Kang Zhu and Dr. Yanfei Mao: pSK-AtU6-sgR, a construct

with the Arabidopsis thaliana small nuclear RNA U6 gene

promoter (AtU626) and restriction sites for cloning in single

guide RNAs (sgRNAs), and p35S-Cas9-SK, a construct with a

human codon-optimized S. pyogenes Cas9 (hSpCas9)

sequence under the control of a double CMV 35S promoter.

For each construct, we ligated in a pair of oligos with the

desired RNA sequence into pSK-AtU6-sgR. Then, using

standard restriction digestion and ligation we generated

constructs with the guide RNAs and the Cas9 nuclease in our

desired plant-expression backbone.

Construct with a single guide RNA

Construct with two guide RNAs to induce a larger deletion

Empty vector control construct

CRISPR-Cas nuclease mutagenesis for genetic containment of

genetically engineered forest treesEstefania Elorriaga, Amy L. Klocko, Cathleen Ma, and Steven H. StraussDepartment of Forest Ecosystems and Society, Oregon State University, Corvallis, OR 97331, USA

AbstractGene flow from GE trees into feral or wild populations are significant obstacles to their use as a result of regulatory, public perception, and ecological concerns. Because many intensively cultivated trees are vegetatively propagated, inducing complete sexual sterility is a

powerful means to reduce these concerns. Loss-of-function mutations in essential floral genes have led to complete or nearly complete sterility in many plant species, however such mutations are rare and generally recessive, thus are very difficult to use via conventional tree

breeding. The recent development of efficient systems for site-directed mutagenesis, particularly the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated Cas system, appear to be powerful tools for generating homozygous loss-of-function

mutations in diploid or even polyploid species. Compared to other sterility methods, the mutations the CRISPR Cas system induces should be highly predictable and stable, as reversion will be extremely rare or impossible (e.g., with deletions of essential parts of coding

regions). We are testing the mutation efficiency of six CRISPR-Cas nucleases targeting two essential floral genes in poplar (Populus). The targets are the poplar orthologs of LEAFY and AGAMOUS from Arabidopsis thaliana. We produced six CRISPR and one empty vector

control (Cas9 only) construct and produced approximately 120 transgenic shoots per construct. Analyses of approximately one-quarter of this population indicate the LEAFY CRISPR-Cas nucleases have an average biallelic mutation efficiency of ~34%, and that major

deletions and rearrangements can be readily identified.

CRISPR system is highly effective for poplar mutagenesis Example of mutations: Deletions (top ) and insertions (bottom)

Single LFY1C (protospacer sequence is in yellow)

Single LFY3C

Double LFY1C-LFY3C

Summary

• CRISPR-Cas nucleases are highly effective at inducing site-directed mutations at the target loci in poplar.

• The majority of the mutations occur 3bp upstream from the protospacer adjacent motif (PAM) site as expected.

• Constructs with a single guide RNA lead generally to small indel mutations.

• Construct with two guide RNAs targeting the same locus generally lead to large deletions.

• Transgenic controls with only the Cas9 gene show wildtype sequence to date.

• The CRISPR-Cas is appears to be a highly effective mutagenesis method in poplar.

pK2GW7LB RBnptII

sgRNA hCas9AtU6-26 2x35S tnos

pK2GW7

sgRNA 1AtU6-26

LB RBnptII

sgRNA 2 hCas9AtU6-26 2x35S tnos

pK2GW7

LB RBnptII

hCas92x35S tnos

Selection of target genes based on highly suppressed RNAi phenotypes

In our attempt to generate

sterile poplar, we have

chosen the poplar

homologs to the

Arabidopsis thaliana LFY

and AG genes; PtLFY and

PtAG. The photos to the

left belong to the events

with the most dramatic

phenotypes from a large

RNA interference (RNAi)

field study. We expect that

some of the CRISPR-Cas

nuclease mutants will have

even more striking, fully

sterile phenotypes.

Construct GE events sequenced Type of mutation # of events (%)

Single LFY1C 92

Bi-allelic INDEL 36 (39%)

Mosaic 42 (46%)

None 14 (15%)

Single LFY3C 46

Bi-allelic INDEL 16 (35%)

Mosaic 27 (58%)

None 3 (7%)

Double LFY1C-

LFY3C59

Bi-allelic INDEL 15 (25%)

Mosaic 40 (68%)

None 4 (7%)

Cas (empty vector) 6 None 6 (100%)

Total (w/out control) 197 Bi-allelic INDEL 67 (34%)

Project overview

Build constructsTransform poplar

tissue with Agrobacterium

Grow transformed

plantlets

Extract DNA and gel-purify gene

amplicons

Sequence amplicons

across target sites

Identify mutation types and determine frequency

• Selected target genes for mutagenesis

• Designed and assembled CRISPR-Cas

nuclease constructs including an empty

vector control

• Produced stably transformed plants using

Agrobacterium tumefaciens

• Isolated DNA from regenerated plantlets

• Extracted DNA from rooted events

• PCR-amplified across target sites

• Aligned the sequences to determine mutant

prevalence and mutation typesSteps for CRISPR-Cas mutagenesis in poplar flowering genes

AcknowledgementsWe thank the industrial members of the Tree Biosafety and Genomics Research Cooperative (TBGRC) at Oregon State University for

support of this project. We also thank the United States Department of Agriculture (USDA, award 2011-68005-30407, System For Advanced

Biofuels Production From Woody Biomass In The Pacific Northwest), the USDA Biotechnology Risk Assessment (grants 2011-68005-30407

and 2010-33522-21736), NSF I/UCRC Center for Advanced Forestry (grant 0736283), USDA-IFAS (grant OREZ-FS-671-R), the Schmidt

Foundation, and the TBGRC industrial cooperative for their support with the RNAi field study. We thank Dr. Jian‐Kang Zhu at Purdue

University and Dr. Yanfei Mao at Shanghai Center for Plant Stress Biology for providing the Strauss lab with the CRISPR-Cas constructs.

We thank Sarah Robertson for the early flowering poplar photos. We thank the OSU Graduate School for travel funding. Last, but not least,

we thank the undergrad workers Analeslie Martinez and Melissa Meyhoff for all their hard work in the laboratory.

Deletion of repressor binding site

A LFY3C mutant has a 22-bp deletion in the promoter region of

PtLFY that completely removed what appears to be the GAGA

motif. This site is known to play a role in recruitment of

polycomb group proteins during development in plants. LFY

might have its expression repressed by polycomb group

proteins prior to flower formation. Its removal might thus

accelerate onset of flowering.

x3

x2

x2

x8

x1

x1

x14

x12

x6

x4

x1

x14

x6

x5

x3

x1

x1

Gel image of 391 bp

insertion in two different

events with LFY1C. This

deletion is not in the

alignment because of its

large size.

H2O

WT

Independent

events

Retransformation with AtFT for early flowering

Poplars trees take between four and five years to reach sexual maturity in the wild,

greatly impeding research on transgenic modification of flowering. We have tested

numerous constructs using the Flowering Locus T (AtFT) gene or its poplar homologs,

to induce precocious flowering. We will retransform various CRISPR-Cas nuclease

mutants with an AtFT constructs to accelerate study flowering phenotypes.

Constructs tested: p409S::AtFT, Han::PCEN + p409S::AtFT, HSP::AtFT, and Han::PCEN + HSP::AtFT.

x4

x1

X1

x9

x4

x3

Double CRISPR leads to inversion

Four LFY1C-LFY3C mutants have an inversion between the

two target sites.

>PtLFY_wildtypeCCAGTGAAAGATCACAGAGAGAGAGACAAGGGGGCAGATAGATATGGATCCGGAGGCTTTCACGGCGAGTTTGTTCAAATGGGACACGAGAGCAATGGTGCCACATCCTAACCGTCTGCTTGAAATGGTGCCCCCGCCTCAGCAGCCACCGGCTGCGGCGTTTGCTGTAAGGCCAAGGGAGCTATGTGGGCTAGAGGAGTTGT

>PtLFY_mutant_LFY1CLFY3C_17CCAGTGAAAGATCACAGAGAGAGAGAGCTGCTGAGGCGGGGGCACCATTTCAAGCAGACGGTTAGGATGTGGCACCATTGCTCTCGTGTCCCATTTGAACAAACTCGCCGTGAAAGCCTCCGGATCCATATCTATCTGCCCCCTTGCACCGGCTGCGGCGTTTGCTGTAAGGCCAAGGGAGCTATGTGGGCTAGAGGAGTTGTTTCA

H2O

WT

Independent events

pla

sm

id

mosaic

mosaic

mosaic

mosaic

mosaic

lgd

el

no

ne

Example of large deletion and mosaicism

Gel image to the left shows seven LFY1C-LFY3C

independent events with different mutation types across

the two alleles: five mosaics (deletion and/or insertion in

one allele or both), one biallelic large deletion, and one

non-mutant.

>PtLFY_wildtypeGAATTCGCCCTTCCTGTTAAGGGCAGTTTTGGTATATAAATAAACAAGAAGCTCACTTGTCTTTATATATCTACCAAATCCAAGACATGCACCAGTGAAAGATCACAGAGAGAGAGACAAGGGGGCAGATAGATATGGATCCGGAGGCTTTCACGGCGAGTTTGTTCAAATGGGACACGAGAGCAATGGTGCCACATCCTAACCGTCTGCTTGAAATGGTGCCCCCGCCTCAGCAGCCACCGGCTGCGGCGTTTGCTGTAAGGCCAAGGGAGCTATGTGGGCTAGAGGAGTT

Example of large insertion

No mutation in controls

Gel image showing the

six Cas (empty vector)

independent events that

have been sequenced so

far. No obvious

mutations seen in gel.

H2

O

WT

Independent

events

pla

sm

id