Generation of Hematopoietic Stem Progenitor Cell Specific Reporter in Human iPSC

Casey Patch, Michael Chen, Thorsten Schlaeger, George Daley

Wikipedia

Structure Applications

(Nat Methods. 2013 Oct; 10(10): 957–963.)

CRISPR

Homology Directed Repair

https://www.addgene.org/CRISPR/guide/

Right Arm Region

gRNA 3’ UTR

Endogenous

Coding Region (ORF)

Edited

Right Arm RegionLeft Arm Region H2B-mKO2

T2A

Schematic Targeting Plan

HomologousRecombination

Left Arm Region Genome

Targeting/Cloning PlanTargeting 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

ORF

VWRPY motifgRNA

Synthesized DNAIncluding 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

ddPCR Information

(Biorad Droplet Digital PCR Applications Guide, 2014)

(A) (B)

(C)

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.