Post on 29-May-2020
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