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Efficient single-copy HDR by 5’ modified long dsDNA donors

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Cite this article as: eLife 2018;7:e39468 doi: 10.7554/eLife.39468
3 figures, 1 table and 3 additional files

Figures

Figure 1 with 1 supplement
Modification of 5’ ends of long dsDNA fragments prevents in vivo multimerization.

(A) Schematic representation of long dsDNA donor cassette PCR amplification with universal primers (black arrows) complementary to the cloning vector backbone outside of the assembled donor cassette (e. g. gfp with homology flanks). Bulky moieties like Biotin at the 5’ ends of both modified primers (red octagon) prevent multimerization/NHEJ of dsDNA, providing optimal conditions for HDR-mediated single-copy integration following CRISPR/Cas9-introduced DSB at the target locus (grey scissors). Representation of locus (Lf/Lr) and internal gfp (Gf/Gr) primers for PCR genotyping of putative HDR-mediated gfp integration events. (B) Southern blot analysis reveals the monomeric state of injected dsDNA fragments in vivo for 5’ modification with Biotin or Spacer C3. Long dsDNAs generated with control unmodified primers or Amino-dT attached primers multimerize as indicated by a high molecular weight ladder apparent already within two hours post-injection (hpi). Note: 5’ moieties did not enhance the stability of injected DNA.

https://doi.org/10.7554/eLife.39468.003
Figure 1—figure supplement 1
Schematic representation of the donor plasmids.

(A–D) Schematic to-scale representation of respective target locus (A, rx2; B, rx1; C, actb; D, dnmt1) with UTR (white boxes with red outlines) and exons (red boxes) highlighted. Homology flanks (HF, grey), sgRNA target sites (white scissors) and respective locus primers (black arrows, Lf, Lr, see Supplementary file 2) are indicated. Respective assembled donor plasmid (A, B, C Golden GATEway (Kirchmaier et al., 2013) or D, conventional cloning) that served as a template for PCR amplification of the unmodified/modified long dsDNA gfp donor cassette is depicted below (FL, flexible linker; backbone sequence in blue). Entry vectors (EV) or restriction enzyme sites for cloning are indicated. The position of primers flanking the donor cassette is indicated, modification highlighted by a red octagon.

https://doi.org/10.7554/eLife.39468.004
Figure 2 with 3 supplements
Modification of 5’ ends of long dsDNA fragments promotes HDR-mediated single-copy integration.

(A) GFP expression in the respective expression domain after HDR-mediated integration of modified dsDNA gfp donor cassettes into rx2, rx1, actb and dnmt1 ORFs in the injected generation. (B) Individual embryo PCR genotyping highlights efficient HDR-mediated single-copy integration of 5’Biotin modified long dsDNA donors, but not unmodified donor cassettes. Locus PCR reveals band size indicative of single-copy gfp integration (asterisk) besides alleles without gfp integration (open arrowhead). Amplification of gfp donor (white arrow) for control.

https://doi.org/10.7554/eLife.39468.005
Figure 2—figure supplement 1
Quantification of survival and GFP expression of injected embryos.

Embryos injected with unmodified or 5’Biotin, Amino-dT or Spacer C3 modified long dsDNA gfp-rx2 donor cassettes were scored for survival at one dpi, and for GFP expression at two dpi. n, total number of injected embryos.

https://doi.org/10.7554/eLife.39468.006
Figure 2—figure supplement 1—source data 1

Quantification of survival and GFP expression of injected embryos.

https://doi.org/10.7554/eLife.39468.007
Figure 2—figure supplement 2
5’Biotin modification of long dsDNA donors strongly enhances HDR-mediated integration.

(A) rx2 locus PCR genotyping of individual GFP-Rx2 positive embryos injected with unmodified or 5’Biotin or Spacer C3 modified long dsDNA gfp donor cassettes (green asterisk, single-copy HDR-mediated integration of gfp, 2547 bp, open arrowhead, rx2 allele without gfp integration, 1719 bp;). Horizontal bar, individual embryo; L, rx2 locus PCR with rx2 Lf/rx2 Lr; G, gfp internal PCR for control with Gf/Gr. (B) Qualitative summary of band spectrum (single-copy HDR-mediated gfp integration, rx2 allele without gfp integration, other) resulting from PCR genotyping in (A). n, number of genotyped GFP-Rx2 expressing embryos.

https://doi.org/10.7554/eLife.39468.008
Figure 2—figure supplement 3
Stable germline transmission of the single-copy HDR-mediated precise gfp integration.

(A) Schematic to-scale representation of the gfp-rx2 locus with UTR (white boxes with red outlines), exons (red boxes) and homology flanks (HF, grey) highlighted. (B, C) Individual GFP-Rx2 expressing embryos (F0, B; F1, C) were genotyped using primers rx2 Lf/rx2 Lr (black arrows) and sequenced with gfpf and gfpr primers (green arrows).

https://doi.org/10.7554/eLife.39468.009
Figure 3 with 1 supplement
Single-copy integration of long dsDNA donor establishes stably transmitted gfp-rx2 fusion gene.

(A) F2 homozygous embryos exhibit GFP-Rx2 fusion protein expression in the pattern of the endogenous gene in the retina. (B) Southern Blot analysis of F2 gfp-rx2 embryos reveals a single band for a digestion scheme cutting outside the donor cassette (BglII/HindIII) or within the 5’ donor cassette and in intron 2 (ScaI/HindIII) indicating precise single-copy donor integration. (B’) Schematic representation of the modified locus indicating the restriction sites and the domain complementary to the probe used in (B). (C) RT-PCR analysis on mRNA isolated from individual homozygous F3 embryos indicates the transcription of a single gfp-rx2 fusion mRNA in comparison to the shorter wild-type rx2 mRNA as schematically represented in (C’).

https://doi.org/10.7554/eLife.39468.010
Figure 3—figure supplement 1
Stably transmitted single-copy integration of the gfp-rx1 donor cassette.

(A) Southern Blot analysis of F2 gfp-rx1 embryos reveals a single band for a digestion scheme cutting outside the donor cassette and within the 3’ donor cassette (HindIII/XmaI) or within the 5’ donor cassette and in intron 1 (NcoI/EcoRI) indicating precise single-copy donor integration. (A’) Schematic representation of the modified locus indicating the restriction sites and the domain complementary to the probe used in (A).

https://doi.org/10.7554/eLife.39468.011

Tables

Key resources table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional information
Strain, strain
background
(Oryzias latipes)
Cabothermedaka Southern wild-type population
Strain, strain
background
(Oryzias latipes)
rx2-gfpthis paper
Strain, strain
background
(Oryzias latipes)
rx1-gfpthis paper
Strain, strain
background
(Oryzias latipes)
actb-gfpthis paper
Strain, strain
background
(Oryzias latipes)
dnmt1-gfpthis paper
Recombinant
DNA reagent
rx2-gfp donor cassettethis paper
Recombinant
DNA reagent
rx1-gfp donor cassettethis paper
Recombinant
DNA reagent
actb-gfp donor cassettethis paper
Recombinant
DNA reagent
dnmt1-gfp donor cassettethis paper
Sequence-
based reagent
rx2 5'HF fthis paperwith BamHI restriction
site: GCCGGATCCAAGCATGTCAAAACGTAGAAGCG
Sequence-
based reagent
rx2 5'HF rthis paperwith KpnI restriction site:
GCCGGTACCCATTTGGCTGTGGACTTGCC
Sequence-
based reagent
rx2 3'HF fthis paperwith BamHI restriction site:
GCCGGATCCCATTTGTCAATGGAC
ACGCTTGGGATGGTGGACGAT
Sequence-
based reagent
rx2 3'HF rthis paperwith KnpI restriction site:
GCCGGTACCTGGACTGGACTGGAAGTTATTT
Sequence-
based reagent
rx2 sgRNA fthis papersubstituted nucleotides to facilitate T7
in vitro transcription of the sgRNA
oligonucleotides are shown in small letters
TAgGCATTTGTCAATGGATACCC
Sequence-
based reagent
rx2 sgRNA rthis paperAAACGGGTATCCATTGACAAATG
Sequence-
based reagent
rx2 Lf/5’UTRfthis paperTGCATGTTCTGGTTGCAACG
Sequence-
based reagent
rx2 Lrthis paperAGGGACCATACCTGACCCTC
Sequence-
based reagent
actb 5’HF fthis paperwith BamHI restriction site:
GGGGATCCCAGCAACGACTTCGCACAAA
Sequence-
based reagent
actb 5’HF rthis paperwith KnpI restriction site:
GGGGTACCGGCAATGTCATCATCCATGGC
Sequence-
based reagent
actb 3’HF fthis paperwith BamHI restriction site:
GGGGATCCGACGACGATATAGCTG
CACTGGTTGTTGACAACGGATCTG
Sequence-
based reagent
actb 3’HF rthis paperwith KnpI restriction site:
GGGGTACCCAGGGGCAATTCTCAGCTCA
Sequence-
based reagent
actb sgRNA fthis paperTAGGATGATGACATTGCCGCAC
Sequence-
based reagent
actb sgRNA rthis paperAAACGTGCGGCAATGTCATCAT
Sequence-
based reagent
actb Lfthis paperGTCCGAGTTGAGGGTGTCTG
Sequence-
based reagent
actb Lrthis paperCATGTGCTCCACTGTGAGGT
Sequence-
based reagent
dnmt1 5’HF fthis paperwith SalI restriction site:
AATTTGTCGACGCTTTGA
CAGTTAACCTACACG
Sequence-
based reagent
dnmt1 5’HF rthis paperwith AgeI restriction site:
AATTTACCGGTCGTAACTGCA
AACTAAAAAATAAAAC
Sequence
-based reagent
dnmt1 3’HF fthis paperwith SpeI restriction site:
AATTTACTAGTATGCCATCCAGAA
CGTCCTTATCTCTACCAGACGATG
TCAGAAAAAGGTAC
Sequence-
based reagent
dnmt1 3’HF rthis paperwith NotI restriction site:
AATTTGCGGCCGCCTACACATA
TTGTCTGTGATAC
Sequence-
based reagent
mgfpfthis paperwith AgeI restriction site:
AATTTACCGGTACTAGTACCATG
AGTAAAGGAGAAGAACTTTTCAC
Sequence-
based reagent
mgfprthis paperwith SpeI restriction site:
AATTTACTAGTCGCGGCTGCACTT
CCACCGCCTCCCGATCCGCCACC
GCCAGAGCCACCTCCGCCTGAAC
CGCCTCCACCGCTCAGGCTAGCTT
TGTATAGTTCATCCATGCCATG
Sequence-
based reagent
dnmt1 sgRNA fthis papersubstituted nucleotides to
facilitate T7 in vitro
transcription of the sgRNA
oligonucleotides are shown
in small letters
TAgGACATCGTCTGGCAAAGAC
Sequence-
based reagent
dnmt1 sgRNA rthis paperAAACGTCTTTGCCAGACGATGT
Sequence-
based reagent
dnmt1 Lfthis paperCTCAATGTAAACACTTCGTGTCGCTTC
Sequence
-based reagent
dnmt1 Lrthis paperTTGCATGCATATTCAAAGTTGTCAAAG
Sequence-
based reagent
rx1 5’HF fthis paperwith BamHI restriction site:
GCCGGATCCGCATCCGAAAGG
TAAGGACTGCAAACC
Sequence-
based reagent
rx1 5’HF rthis paperwith KpnI restriction site:
GCCGGTACCCATGAGAGCG
TCTGGGCTCTGACC
Sequence-
based reagent
rx1 3’HF fthis paperwith BamHI restriction site:
GGCGGATCCCATTTATCAC
TCGATACCATGAGCA
Sequence-
based reagent
rx1 3’HF rthis paperwith KpnI restriction site:
GGCGGTACCTTCCAGTTTA
AGAACATCCCCTCT
Sequence-
based reagent
rx1 sgRNA1 fthis papersubstituted nucleotides to
facilitate T7 in vitro
transcription of the sgRNA
oligonucleotides are shown
in small letters
TAggAAATGCATGAGAGCGTCT
Sequence-
based reagent
rx1 sgRNA1 rthis paperAAACAGACGCTCTCATGCATTT
Sequence-
based reagent
rx1 sgRNA2 fthis papersubstituted nucleotides to
facilitate T7 in vitro
transcription of the sgRNA
oligonucleotides are shown
in small letters
TAggCTCTCATGCATTTATCAC
Sequence-
based reagent
rx1 sgRNA2 rthis paperAAACGTGATAAATGCATGAGAG
Sequence-
based reagent
rx1 Lfthis paperCTTTGCTGTTTTGAGAATTGCACC
Sequence-
based reagent
rx1 Lrthis paperGAGACCGAACGATGACAATAACAC
Sequence-
based reagent
pDest f (control)this paperCGAGCGCAGCGAGTCAGTGAG
Sequence-
based reagent
pDest r (control)this paperCATGTAATACGACTCACTATAG
Sequence-
based reagent
pDest f modthis paperAsterisks indicate phosphorothioate
bonds, ‘5’moiety’ was either
5’Biotin, Amino-dT or Spacer C3.
5’moiety-C*G*A*G*C*GCAGCGAGTCAGTGAG
Sequence-
based reagent
pDest r modthis paperAsterisks indicate phosphorothioate
bonds, ‘5’moiety’ was either 5’Biotin
, Amino-dT or Spacer C3.
5’moiety-C*A*T*G*T*AATACGACTCACTATAG
Sequence-
based reagent
pCS2 fthis paperCCATTCAGGCTGCGCAACTG
Sequence-
based reagent
pCS2 rthis paperCACACAGGAAACAGCTATGAC
Sequence
-based reagent
pCS2 f modthis paperAsterisks indicate phosphorothioate
bonds, ‘5’moiety’ was either
5’Biotin, Amino-dT or Spacer C3.
5’moiety-C*C*A*T*T*CAGGCTG
CGCAACTG
Sequence-
based reagent
pCS2 r modthis paperAsterisks indicate phosphorothioate
bonds, ‘5’moiety’ was either
5’Biotin, Amino-dT or Spacer C3.
5’moiety-C*A*C*A*C*AGGAAACAGCTATGAC
Sequence-
based reagent
Gfthis paperATGGCAAGCTGACCCTGAAGTTCAT
CTGCACCACCGGCAAGC
Sequence-
based reagent
Grthis paperCTCAGGTAGTGGTTGTCG
Sequence-
based reagent
gfpfthis paperGCTCGACCAGGATGGGCA
Sequence-
based reagent
gfprthis paperCTGAGCAAAGACCCCAACGAGA
AGCGCGATCACATG
Sequence-
based reagent
gfp probe fthis paperGTGAGCAAGGGCGAGGAGCT
Sequence-
based reagent
gfp probe rthis paperCTTGTACAGCTCGTCCATG

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting files. Source data files have been provided for Figure 2-figure supplement 1.

Additional files

Supplementary file 1

Analysis of injected embryos.

Embryos injected with unmodified or modified (5’Biotin, Amino-dT, Spacer C3) long dsDNA gfp donor cassettes matching the rx2, actb, dnmt1 or rx1 locus, were scored for GFP expression and survival. Injections without Cas9 mRNA for control.

https://doi.org/10.7554/eLife.39468.012
Supplementary file 2

Oligonucleotides used in this work.

Restriction enzyme sites used for cloning of PCR amplicons are indicated in italics. Substituted nucleotides to facilitate T7 in vitro transcription of the sgRNA oligonucleotides are shown in small letters (Stemmer et al., 2015). Locus primers forward (Lf) and reverse (Lr) of respective gene loci, gfp primers forward (Gf) and reverse (Gr), gfp sequencing primers gfpf and gfpr and primers to amplify the mgfp-flexible linker, as well as the gfp probe for Southern Blot analysis, are given. Asterisks indicate phosphorothioate bonds, ‘5’moiety’ was either 5’Biotin, Amino-dT or Spacer C3 in the pDest f mod, pDest r mod, pCS2 f mod and pCS2 r mod primers.

https://doi.org/10.7554/eLife.39468.013
Transparent reporting form
https://doi.org/10.7554/eLife.39468.014

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