The Center for Synthetic Regulatory Genomics (SyRGe)

www.thedarkmatterproject.org

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Institute for Systems Genetics

NYU Langone Health

2026 GENOME WRITING WORKSHOP

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Session 3

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Ran Brosh

The Dark Matter Project Genome Writing Pipeline (simplified)

Resources – Our shared Google Doc

I will be switching a lot between:

• This presentation
• SnapGene
• The UCSC Browser
• The Google Doc

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If I’m going too fast, use the slowdown option in the Zoom React menu

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Have a question? Use

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All resources (PPTs, SnapGene files etc.)

Our design goal – complete humanization of mouse Hprt1

Today’s tasks:

1. Identify the boundaries of human HPRT1 to be cloned and delivered
2. Design a MC2 acceptor vector for hHPRT1
3. Generate BED files for:
1. HAs (previous session and new ones)
2. Assembly region
4. Review lambda Red assembly strategy
5. Design a PCR genotyping strategy to identify candidate clones
6. Review delivery timeline and tips
7. Discuss alternative scenarios:
• Scar removal
• Yeast assembly from BACs
• Yeast assembly from fragments
8. Discuss Session 4

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Payload boundaries

How to select the human HPRT1 boundaries?

1. Conservation (mouse/human)
1. UCSC Browser
2. Vista Browser
2. Regulation
3. Neighboring genes
4. GC content
5. Repeats/complexity
6. RE enzymes (SalI)

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Biology

Technical

MC2v2 acceptor vector

Barriball et al., Improved vector toolkit for genome writing in mammalian cells. bioRxiv. 2026.

Our design goal – complete humanization of mouse Hprt1

Today’s tasks:

1. Identify the boundaries of human HPRT1 to be cloned and delivered
2. Design a MC2 acceptor vector for hHPRT1:
1. HAL/HAR for delivery
2. AAL/AAR for assembly
3. Primers and gBlocks
3. Generate BED files for:
• HAs (previous session and new ones)
• Assembly region
4. Review lambda Red assembly strategy
5. Design a PCR genotyping strategy to identify candidate clones
6. Review delivery timeline and tips
7. Discuss alternative scenarios:
• Scar removal
• Yeast assembly from BACs
• Yeast assembly from fragments
8. Discuss Session 4

Gibson assembly

https://www.snapgene.com/guides/gibson-assembly

Our design goal – complete humanization of mouse Hprt1

Today’s tasks:

1. Identify the boundaries of human HPRT1 to be cloned and delivered
2. Design a MC2 acceptor vector for hHPRT1:
1. HAL/HAR for delivery
2. AAL/AAR for assembly
3. Primers and gBlocks
3. Generate BED files for:
• HAs (previous session and new ones)
• Assembly region
4. Review lambda Red assembly strategy
5. Design a PCR genotyping strategy to identify candidate clones
6. Review delivery timeline and tips
7. Discuss alternative scenarios:
• Scar removal
• Yeast assembly from BACs
• Yeast assembly from fragments
8. Discuss Session 4

Our design goal – complete humanization of mouse Hprt1

Today’s tasks:

1. Identify the boundaries of human HPRT1 to be cloned and delivered
2. Design a MC2 acceptor vector for hHPRT1:
1. HAL/HAR for delivery
2. AAL/AAR for assembly
3. Primers and gBlocks
3. Generate BED files for:
• HAs (previous session and new ones)
• Assembly region
4. Review lambda Red assembly strategy
5. Design a PCR genotyping strategy to identify candidate clones
6. Review delivery timeline and tips
7. Discuss alternative scenarios:
• Scar removal
• Yeast assembly from BACs
• Yeast assembly from fragments
8. Discuss Session 4

Lambda Red assembly (LRA) vs yeast assembly

Gam: Prevents linear DNA digestion.

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Exo: Degrades DNA (5’->3’) – produces ssDNA 3’ overhangs.

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Beta: Protects ssDNA overhangs, promotes annealing to complementary ssDNA.

LRA

DH10B

Kelly Barriball

&

Weimin Zhang

Transform LR plasmid

Chl+Amp

selection

Transform linearized

assembly vector

KanR

Kan

selection

ChlR

Replica plate to confirm loss of ChlR (BAC) and AmpR (LR plasmid)

Prep delivery-ready payload vector

(~2 weeks inc. sequencing)

KanR

Results are excellent and fast

Colony PCR with: L/R junctions, human internal segments A-H, chloramphenicol resistance gene

Human gene cluster 214 kb

CHL

bKBA9

L

R

A

C

D

F

G

H

Results in a few days, and products is directly ready to transform into mammalian cells

Our design goal – complete humanization of mouse Hprt1

Today’s tasks:

1. Identify the boundaries of human HPRT1 to be cloned and delivered
2. Design a MC2 acceptor vector for hHPRT1:
1. HAL/HAR for delivery
2. AAL/AAR for assembly
3. Primers and gBlocks
3. Generate BED files for:
• HAs (previous session and new ones)
• Assembly region
4. Review lambda Red assembly strategy
5. Design a PCR genotyping strategy to identify candidate clones
6. Review delivery timeline and tips
7. Discuss alternative scenarios:
• Scar removal
• Yeast assembly from BACs
• Yeast assembly from fragments
8. Discuss Session 4

Our design goal – complete humanization of mouse Hprt1

Today’s tasks:

1. Identify the boundaries of human HPRT1 to be cloned and delivered
2. Design a MC2 acceptor vector for hHPRT1:
1. HAL/HAR for delivery
2. AAL/AAR for assembly
3. Primers and gBlocks
3. Generate BED files for:
• HAs (previous session and new ones)
• Assembly region
4. Review lambda Red assembly strategy
5. Design a PCR genotyping strategy to identify candidate clones
6. Review delivery timeline and tips
7. Discuss alternative scenarios:
• Scar removal
• Yeast assembly from BACs
• Yeast assembly from fragments
8. Discuss Session 4

Delivering Big DNAs to Mammalian Cells – Timeline

Adapted from Zhang et al., Nature 2023. PMCID: PMC10632133

Our design goal – complete humanization of mouse Hprt1

Today’s tasks:

1. Identify the boundaries of human HPRT1 to be cloned and delivered
2. Design a MC2 acceptor vector for hHPRT1:
1. HAL/HAR for delivery
2. AAL/AAR for assembly
3. Primers and gBlocks
3. Generate BED files for:
• HAs (previous session and new ones)
• Assembly region
4. Review lambda Red assembly strategy
5. Design a PCR genotyping strategy to identify candidate clones
6. Review delivery timeline and tips
7. Discuss alternative scenarios:
• Scar removal
• Yeast assembly from BACs
• Yeast assembly from fragments
8. Discuss Session 4

Session 4