Near-perfect precise on-target editing of human hematopoietic stem and progenitor cells

  1. Fanny-Mei Cloarec-Ung
  2. Jamie Beaulieu
  3. Arunan Suthananthan
  4. Bernhard Lehnertz
  5. Guy Sauvageau
  6. Hilary M Sheppard
  7. David JHF Knapp  Is a corresponding author
  1. Institut de Recherche en Immunologie et en Cancérologie, Université de Montréal, Canada
  2. School of Biological Sciences, Faculty of Science, University of Auckland, New Zealand
  3. Département de Pathologie et Biologie Cellulaire, Université de Montréal, Canada
4 figures, 1 table and 6 additional files

Figures

Figure 1 with 2 supplements
AAV and short ssODN both allow precise editing in human hematopoietic stem and progenitor cells (HSPCs).

(A) Experimental design. (B) Homology-directed repair (HDR) donor configurations. The SRSF2 P95H AAV donor is shown above and short and long ssODN donors are shown below with features indicated. Annotated sequences are shown in Supplementary file 2. (C) HDR integration efficiency by AAV dose. Cells were edited with 30.5 pmol ribonuclear protein (RNP) (or not as indicated) with indicated multiplicities of infection (MOI) of AAV donor. Bars show mean values and points show measurements for individual cords. Male cords are shown as triangles and females as circles. (D) Viable cell number by AAV dose. Hemocytometer counts at the time of harvest are shown for each sample from (C). (E) HDR integration efficiency for short and long ssODN donors. Donor DNA amounts are shown in pmol. (F) Viable cell number by ssODN dose. False-discovery rate (FDR) corrected paired t-test significance values are shown in Supplementary file 1. See also Figure 1—figure supplements 1 and 2.

Figure 1—figure supplement 1
Ribonuclear protein (RNP) efficiency determination.

Cells were edited with indicated amounts of Cas9 RNP and 1 x IDT electroporation enhancer where indicated. Cutting efficiencies as measured by T7E1 are shown in (A) for SRSF2 and (C) for SF3B1, and number of viable cells in (B) for SRSF2 and (D) for SF3B1. False-discovery rate (FDR) corrected unpaired t-test significance values are shown in Supplementary file 1.

Figure 1—figure supplement 2
Assays for the detection of homology-directed repair (HDR) integration.

(A) Nested PCR is required to avoid non-specific amplification. The upper gel shows that even in the absence of ribonuclear protein (RNP), single primer pairs amplify AAV from the donor. The lower gel demonstrates that with nested PCR this is no longer the case. (B) Enzymatic digestion can detect editing down to a minority of ~1%. Known proportions of mutant and wild-type PCR products were mixed, digested with BspEI, and the gels quantified. An example gel is shown on the right, and the quantifications from two independent replicates shown on the left. A perfect measure is shown as a blue line and the linear fit in red. (C) Sanger sequencing-based detection of editing. An example chromatogram from an unedited control, a silent edited sample, and a P95H with spacer-breaking silent mutations edited sample are shown. Locations of mutant bases are indicated with a red box for the location of the P95H mutation, and blue boxes for each of the silent mutations. Editing efficiencies calculated by ICE are indicated for each sequence.

Figure 2 with 3 supplements
Small molecule-mediated inhibition of DNA-PK and optimal donor design substantially improve precise editing efficiency.

(A) AZD7648 and M3814 improve homology-directed repair (HDR) efficiency in primary human hematopoietic stem and progenitor cell (HSPC). Cells were edited with 30.5 pmol ribonuclear protein (RNP) (or not as indicated) with 400 multiplicity of infection (MOI) of AAV donor and small molecules added as indicated (in µM). Bars show mean values and points show measurements for individual cords. Male cords are shown as triangles and females as circles. (B) Viable cell numbers with AZD7648 and M3814 addition. Hemocytometer counts at the time of harvest are shown for each sample from (A). (C) HDR efficiency with combinations of AZD7648, p53 siRNA, and RS-1. Cells were edited with 30.5 pmol RNP (or not as indicated) with 400 MOI of AAV donor in the presence of the indicated additives. AZD7648 was used at 5 µM, p53 siRNA at 20 fmol, and RS-1 at 15 µM. (D) Viable cell numbers with additive combinations. Hemocytometer counts at the time of harvest are shown for each sample from (C). (E) Technical factors associated with high sample number is associated with decreased HDR efficiency. HDR efficiency is shown for all 30.5 pmol RNP, 400 MOI AAV, 20 fmol p53 siRNA, and 5 µM AZD7648 samples by the number of conditions processed in a given experiment. A linear fit is indicated as a red line. The R2 is indicated, and overall p-value was <<0.001. (F) Alternative designs for ssODN donors with key features indicated. Annotated sequences are shown in Supplementary Information. (G) Silent mutations allow ssODN donors to achieve similar efficiencies to AAV. All edits were performed with 0.5 µM AZD7648, 20 fmol p53 siRNA, 50 pmol ssODN, or 400 MOI AAV as indicated. Donor types are shown as their logos from (1B, 2F). (H) No observable off-target mutations at predicted target sites even with the addition of AZD7648. The overall percent of reads containing exclusively reference allele, or any substitutions, deletions, or insertions that overlap with the predicted off-target cut sites is shown for three individual cords across the top 3 cut sites. Cells from each individual cord were split into an unedited control, and cells edited with the silent mutation containing ssODN for the SRSF2 locus under either standard conditions (i.e. no p53siRNA or AZD7648) or with our optimal editing protocol (i.e. with p53siRNA and 0.5 µM AZD7648). False-discovery rate (FDR) corrected paired t-test significance values are shown in Supplementary file 1. See also Figure 2—figure supplements 13.

Figure 2—figure supplement 1
The addition of AZD7648 also improves editing efficiency at the SF3B1 locus.

(A) Homology-directed repair (HDR) efficiency for SF3B1 K700E with combinations of AZD7648, p53 siRNA and RS-1. Cells were edited with 30.5 pmol ribonuclear protein (RNP) (or not as indicated) with 400 multiplicity of infection (MOI) of AAV donor in the presence of the indicated additives. AZD7648 was used at 5 µM, p53 siRNA at 20 fmol, and RS-1 at 15 µM. (B) Viable cell numbers with additive combinations. Hemocytometer counts at the time of harvest are shown for each sample from (A). False-discovery rate (FDR) corrected unpaired t-test significance values are shown in Supplementary file 1.

Figure 2—figure supplement 2
Example Sanger sequencing traces for the top three predicted off-target sites of the SRSF2 gRNA.

Chromosome number and location displayed are indicated for each site. Dotted lines indicate the expected recognition site based on predictions from Benchling. Off-target site 2 is displayed from the reverse strand. All traces were analyzed using ICE and had an estimated cutting of 0% with R2 values between 0.99 and 1. One example chromatogram for each site is shown, however, three independent donors were analyzed for each. Trace views were exported using Chromas.

Figure 2—figure supplement 3
Full-length nanopore sequencing traces for the top three predicted off-target sites of the SRSF2 gRNA.

(A) Frequency of each base type across off-target amplicons. Chromosome number and location are displayed above each column of samples with the overall amplicon first and the specific predicted target location in brackets. The frequency of reads at a given base that were either reference allele, a substitution (from reference), an insertion, or a deletion are shown for each base across the amplicon. Only reads with a Phred score of at least 16 at a given base were included. Results are shown for unmanipulated control, standard editing (ie, no AZD7648), and edited with our optimal protocol (including AZD7648) are shown for each of three individual cord donors (Samples 1–3). Of these, two were male (Samples 1 & 2) and one female (Sample 3). (B) Read depth Per Individual across each amplicon. The total read depth of accepted bases (ie. those with a Phred score of at least 16) is shown for each sample and region. Target regions are highlighted in gray.

Figure 3 with 1 supplement
Editing has a minimal impact on hematopoietic stem and progenitor cell (HSPC) function and hierarchy.

(A) Integration efficiency is equivalent across phenotypically defined progenitor compartments. All edits were performed with 0.5 µM AZD7648, 20 fmol p53 siRNA, and 50 pmol of silent mutation ssODN. Values show the difference in precise edit efficiency for each phenotypic subset compared to bulk assessment within that cord. Bars show mean values and points show measurements for individual cords. Male cords are shown as triangles and females as circles. All populations show no significant difference from bulk. (B) Progenitor phenotypes are minimally altered across the hierarchy. The % of CD34 + for each sub-population is shown. Mean values are indicated as lines. A slight but significant decrease was present for late progenitors (CD34 +CD45RA+) associated with donor addition (but not different with editing). (C) An example image of a well of colonies, and example colonies. (D) Total colonies are decreased by the addition of donors, and further by editing. Total CFC per 1000 CD34 + cells is shown for each cord. Lines indicate mean values. (E) No changes were observed in the frequency of colonies of each type. As before points are individual cords and lines show mean values. (F) Colonies showed a preponderance of homozygous editing. Mean homozygous, heterozygous edited, and unedited cells are shown from 36 analyzed colonies across three independent cords. False-discovery rate (FDR) corrected paired t-test significance values are shown in Supplementary file 1. (G) No change in the dynamics of colony emergence from single-LT-HSCs in long-term culture initiating cell (LTC-IC). The presence or absence of an obvious colony in each well (initially sorted with a single long-term HSC (LT-HSC)) was scored weekly over the first 6 weeks of the LTC-IC assay, and again at week 8. Clonal outputs are shown as lines with unedited in black and edited in blue. (H) Example colonies at 8 weeks. At 8 weeks, clones were scored as negative (no colony at any point), transient (previous colony without a colony at endpoint), low proliferation (>50 cells, but below confluence), and highly proliferative (confluent). Example images of negative, low proliferation, and highly proliferative clones are shown. Scalebars (white) show 1 mm. The low proliferation colony is circled in red. (I) Highly proliferative clones are not lost from the LT-HSC population in the editing process. The frequency of clones of the indicated types is shown per 100 phenotypic LT-HSC either without editing or following optimal editing. Error bars represent 95% confidence intervals. Frequencies, p-values, and error bars were calculated using Extreme Limiting Dilution Analysis, based on colony numbers measured from three independent experiments (each with a different cord donor). Numbers for each clone per donor, the total number of clones analyzed for that donor, and donor sex are indicated below the relevant bar. See also Figure 3—figure supplement 1.

Figure 3—figure supplement 1
Example gating hierarchy.

FMO stands for full-minus-one, where the indicated antibody was left out of the panel.

Zygosity can be tuned using a mixture of mutant and silent donors.

(A) Experimental design. Green bars represent the proportion of mutant donor. Specific amounts of mutant and silent donor are shown underneath for each condition. All edits were performed with 0.5 µM AZD7648, 20 fmol p53 siRNA, and indicated amounts of each donor. (B) Overall mutant integration efficiency varies linearly with the proportion of mutant donor. Individual cords are shown as points. Male cords are shown as triangles and females as circles. A linear fit is indicated as a red line. The R2 is indicated, and overall p-value was <<0.001. (C) Mean clonogenic frequencies are consistent across donor proportions. Data are a mean of two independent cords with a total of 30 cells for each cord at each dose analysed except the 0% condition which only had 20 cells per cord. (D) Zygosity can be adjusted by inclusion of silent donor. Mean frequencies of homozygous mutant, heterozygous, and homozygous silent donors are shown within all clones with any observed editing. A total of 52 clones with some degree of editing across two independent cords were analyzed.

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
AntibodyAF647 (mouse monoclonal) Anti-Human CD34 (clone 581)Cedarlane3435081:200
AntibodyV450 (mouse monoclonal) Anti-Human CD45RA (clone HI100)BD Biosciences5603621:100
AntibodyPE-CF594 (mouse monoclonal) Anti-Human CD90 (clone 5E10)BD Biosciences5623851:200
AntibodyFITC anti-human, CD49c (Clone REA360)Miltenyi130-105-3641:50
Genetic reagent (AAV2/6)Custom AAV6 – pssAAV_SRSF2P95HCanadian Neurophotonics Platform – Viral Vector CoreCustom AAVSee annotated sequences in Supplementary file 2
Genetic reagent (AAV2/6)Custom AAV6 – pssAAV_SF3B1 K700ECanadian Neurophotonics Platform – Viral Vector CoreCustom AAVSee annotated sequences in Supplementary file 2
Biological sample (Homo sapiens)Human Cord Blood for CD34 + cells harvestHéma Québec via St Justine hospitalNA
Cell line (Mus musculus)M210B4 expressing human IL-3 and G-CSFGift from Connie J EavesNA*special request
Cell line (Mus musculus)sl/sl mouse fibroblasts expressin human SCF and IL-3Gift from Connie J EavesNA*special request
Cell line (Mus musculus)sl/sl mouse fibroblasts expressin human FLT3LGift from Connie J EavesNA*special request
Chemical compound, drugUM 171ExcellTheraNA*special request
Chemical compound, drugRS-1Cedarlane21037–5
Chemical compound, drugNedisertib (M3814)CedarlaneA17055
Chemical compound, drugAZD 7648Cedarlane (Cayman)28598–1
Chemical compound, drugDIMETHYL SULFOXIDE (DMSO), SterileBioShopDMS666.100
Chemical compound, drugHydrocortisoneBioShopHYD400.5
Chemical compound, drug1 M bufferHomemadeHomemade
Commercial assay, kitPLATINUM SUPERFI II MASTER MIXLife Technologies12368050
Commercial assay, kitStemSpan CC100STEMCELL Technologies2690
Commercial assay, kitMyeloCult H5100STEMCELL Technologies05150
Commercial assay, kitBlunt/TA Ligase Master MixNEBM0367S
Commercial assay, kitNEBNext Quick Ligation ModuleNEBE6056S
Commercial assay, kitNEBNext Ultra II End Repair/dA-Tailing ModuleNEBE7546S
Commercial assay, kitEasySep Human CD34 Positive Selection Kit IISTEMCELL Technologies17896
Commercial assay, kitMethoCult H4034 OptimumSTEMCELL Technologies4034
Commercial assay, kitFlongle Sequencing ExpansionOxford NanoporeFLO-FLG114
Commercial assay, kitFlongle Flow Cell (R10.4.1)Oxford NanoporeFLO-FLG114
Commercial assay, kitNative Barcoding Kit 24 V14Oxford NanoporeSQK-NBD114.24
Commercial assay, kitFBS CanadienThermo12483020
Commercial assay, kitRPMI1640LifeTech11875119
Commercial assay, kitStemSpan SFEM IISTEMCELL Technologies9655
Peptide, recombinant proteinAlt-R S.p. Cas9 Nuclease V3, 500 µgIDT1081058
Peptide, recombinant proteinT7 Endonuclease I - 250 unitsNEBM0302S
Peptide, recombinant proteinProteinase K, Molecular Biology GradeNEBP8107S
Peptide, recombinant proteinAlt-R Cas9 Electroporation Enhancer, 2 nmolIDT1075915
Peptide, recombinant proteinBspEI EnzymeNEBR0540S
Peptide, recombinant proteinIL-3, Human (CHO-expressed), 100 ng/ulCedarlane (GeneScript)Z02991-10
Peptide, recombinant proteinSCF, Human (P. pastoris-expressed), 100 ng/ulCedarlane (GeneScript)Z02692-10
Peptide, recombinant proteinEPO 100 ng/ul (~16 IU/uL)Cedarlane (GeneScript)Z02975-10
Peptide, recombinant proteinFlt-3L 100 ng/ulCedarlane (GeneScript)Z02926-10
Peptide, recombinant proteinGM-CSF, Human (CHO-expressed), 100 ng/uLCedarlane (GeneScript)Z02983-10
Peptide, recombinant proteinIL-6, Human (CHO-expressed), 100 ng/uLCedarlane (GeneScript)Z03134-50
Peptide, recombinant proteinG-CSF, Human (CHO-expressed), 100 ng/uLCedarlane (GeneScript)Z02980-10
Peptide, recombinant proteinCellAdhere Type I Collagen, Bovine, SolutionSTEMCELL Technologies7001
Recombinant DNA reagentp53 siRNA id s605Thermo4390824
Sequence-based reagentSRSF2_gRNA1IDT/AlTR1/rCrGrGrCrUrGrUrG
rGrUrGrUrGrArGrUrCrCr
GrGrGrUrUrUrUrArGrAr
GrCrUrArUrGrCrU/AlTR2/
crRNA SRSF2
Sequence-based reagentpri0077-FIDTAGCGATATAAACGGGCGCAGOuter PCR SRSF2
Sequence-based reagentpri0077-RIDTTCGCGACCTGGATTTGGATTOuter PCR SRSF2
Sequence-based reagentpri0002-H3IDTCTATGGATGCCATGGACGGGInner PCR SRSF2
Sequence-based reagentpri0002-H4IDTCAAGCACAGCGGGGTTAATTCInner PCR SRSF2
Sequence-based reagentpri0261-FIDTTCATTGGCAAACAGCAAGCCSRSF2 gRNA1 off-target 1
Sequence-based reagentpri0261-RIDTAGAAGTATGTGCCTACGCGGSRSF2 gRNA1 off-target 1
Sequence-based reagentpri0262-FIDTGAGAGTCACCGACCATGACGSRSF2 gRNA1 off-target 2
Sequence-based reagentpri0262-RIDTTGTAAAACGTGCTGGAGGCTSRSF2 gRNA1 off-target 2
Sequence-based reagentpri0263-FIDTCAGAAAGCACAAGCAACGCTSRSF2 gRNA1 off-target 3
Sequence-based reagentpri0263-RIDTTCTCTTCCGGACACAAGTGCSRSF2 gRNA1 off-target 3
Sequence-based reagentpri0285IDTCTCCTTCTTCACGTCTTCCTSRSF2 off-target 2 sequencing
Sequence-based reagentpri0286IDTCACCACATCTGGGATCCTCASRSF2 off-target 3 sequencing
Sequence-based reagentSF3B1 Cas9 gRNA K700IDT/AlTR1/rUrGrGrArUrGrArGrCrArGr
CrArGrArArArGrUrUrGrUrUrUrUrAr
GrArGrCrUrArUrGrCrU/AlTR2/
crRNA SF3B1
Sequence-based reagentAlt-R CRISPR-Cas9 tracrRNAIDT1072533tracrRNA
Sequence-based reagentpri0078-FIDTGCTGCTGGTCTGGCTACTATOuter PCR SF3B1
Sequence-based reagentpri0078-RIDTATACTCATTGCTGATTACGTGATTTOuter PCR SF3B1
Sequence-based reagentpri0002-H1IDTTGGGCTACTGATTTGGGGAGInner PCR SF3B1
Sequence-based reagentpri0002-H2IDTCTGTGTTGGCGGATACCCTTInner PCR SF3B1
Sequence-based reagentSRSF2 Silent ssODNIDTt*g*gacggccgcgagctgcgggtgcaaatg
gcgcgctacggccgcccTccAgaTtcacacca
cagccgccggggaccgccaccccgcag*g*t
Sequence-based reagentSRSF2 P95H ssODNIDTt*g*gacggccgcgagctgcgggtgcaaatggcg
cgctacggccgccATccggactcacaccacag
ccgccggggaccgccaccccgcag*g*t
Sequence-based reagentSRSF2 long ssODN donorIDTTtcacgacaagcgcgacgctgaggacgctatgga
Tgccatggacggggccgtgctggacggccgcga
gctgcgggtgcaaatggcgcgctacgg
ccgccATccggactcacaccacagccgccggg
gaccgccaccccgcaggtacgggggcggtggcta
cggacgccggagccgcaggtaaacgg
ggctgaggggaccg
ordered as ALT-R HDR Donor Oligo
Sequence-based reagentSRSF2 P95H ssODN with additional silent mutationsIDTt*g*gacggccgcgagctgcgggtgcaaatggcgcgctac
ggccgccATccAgaTtcacaccacagccgccggg
gaccgccaccccgcag*g*t
Software and algorithmsGelAnalyzer 19.1Istvan Lazar Jr. and Istvan Lazar Sr.https://www.gelanalyzer.com
Software and algorithmsSynthego Performance Analysis V3ICE Analysishttps://www.synthego.com
Software and algorithmsFlowJo Software 10.8.1BD Life Scienceshttps://www.flowjo.com
Software and algorithmsR (version 4.1.2)R Core Teamhttps://www.r-project.org/
Software and algorithmsMinKNOW (version 23.11.3)Oxford Nanoporehttps://community.nanoporetech.com/downloads
Software and algorithmsminimap2 (version 2.26)Dana-Farber Cancer Institutehttps://github.com/lh3/minimap2
Software and algorithmssamtools (version 1.10)Genome Research Ltd.http://www.htslib.org/
Software and algorithmsbcftools (version 1.10.2)Genome Research Ltd.https://samtools.github.io/bcftools/
Software and algorithmsBiopython (version 1.83)Biopythonhttps://biopython.org/
Software and algorithmsPython (version 3.9.7)Pythonhttps://www.python.org/

Additional files

Supplementary file 1

Significance testing.

Figure and panel are indicated along with each pair-wise test. Where relevant the colony type or population for a given test is indicated.

https://cdn.elifesciences.org/articles/91288/elife-91288-supp1-v1.docx
Supplementary file 2

Annotated DNA sequence used in the experiments for HDR testing and integration.

https://cdn.elifesciences.org/articles/91288/elife-91288-supp2-v1.docx
MDAR checklist
https://cdn.elifesciences.org/articles/91288/elife-91288-mdarchecklist1-v1.docx
Source data 1

Raw gel images.

Data file for the raw gels compiled in Source data 2.

https://cdn.elifesciences.org/articles/91288/elife-91288-data1-v1.zip
Source data 2

Annotated gels.

Compilation of the annotated gels used for quantification of RNP and HDR integration efficiencies.

https://cdn.elifesciences.org/articles/91288/elife-91288-data2-v1.pdf
Source data 3

Numeric data.

Compilation of the numeric data used throughout the manuscript.

https://cdn.elifesciences.org/articles/91288/elife-91288-data3-v1.zip

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  1. Fanny-Mei Cloarec-Ung
  2. Jamie Beaulieu
  3. Arunan Suthananthan
  4. Bernhard Lehnertz
  5. Guy Sauvageau
  6. Hilary M Sheppard
  7. David JHF Knapp
(2024)
Near-perfect precise on-target editing of human hematopoietic stem and progenitor cells
eLife 12:RP91288.
https://doi.org/10.7554/eLife.91288.3