This document describes a targeting plan to generate a hematopoietic stem progenitor cell specific reporter in human induced pluripotent stem cells (iPSCs) using CRISPR/Cas9 genome editing. The plan involves designing gRNAs to target an endogenous coding region. A targeting construct containing homologous arms and a T2A-H2B-mKO2 cassette will be used for homology directed repair. The gRNAs and targeting construct will be transfected into iPSCs and successful homologous recombination can be detected using digital droplet PCR.

1. Generation of Hematopoietic Stem Progenitor Cell
Specific Reporter in Human iPSC
Casey Patch, Michael Chen, Thorsten Schlaeger,
George Daley

2. Wikipedia

4. Structure Applications
(Nat Methods. 2013 Oct; 10(10): 957–963.)
CRISPR

5. Homology Directed Repair
https://www.addgene.org/CRISPR/guide/

6. Right Arm Region
gRNA 3’ UTR
Endogenous
Coding Region (ORF)
Edited
Right Arm RegionLeft Arm Region H2B-mKO2
T2A
Schematic Targeting Plan
Homologous
Recombination
Left Arm Region Genome

8. Targeting/Cloning Plan
Targeting Construct Cloning:
● PCR amplification of left and
right homologous arms
● Synthesized 2A-H2BmKO2
○ Cloning of these
components together
● Verify Sequence
CRISPR:
● Design gRNAs with crispr.mit.
edu CRISPR design tool and
clone following Church Lab’s
gRNA Synthesis Protocol
● Transfect into HEK293T cells
and run ddPCR to test gRNA
efficiency and establish protocol
● Transition to iPS cells and co-
transfect with targeting construct

9. ORF
VWRPY motifgRNA
Synthesized DNA
Including T2A-H2B-mKO2
Left Arm (3 kb) Right Arm (1 kb)15 bp In-Fusion overhang
15 bp In-Fusion overhang
Cloning Strategy
gRNA site with silent mutations

10. ddPCR Information
(Biorad Droplet Digital PCR Applications Guide, 2014)

12. (A) (B)
(C)

13. Acknowledgements
● Thanks to Boston Children’s Hospital and
Harvard Medical School for the opportunity
to research in their facilities. Thanks to
Boston College for everything.
● I would like to thank my PI, Thorsten
Schlaeger. My mentor Michael Chen and
colleagues Manav Gupta, Blair Bell, Alex
Devine, and Mike Shi for all of their help in
my project.
● I would like to thank Dr. Clare O’Conor for
her guidance and discussions as my
research advisor during Junior Year
● Of course, I would like to thank George
Daley for this opportunity and his guidance
in my work.