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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 2

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Ran Brosh

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The Dark Matter Project Genome Writing Pipeline (simplified)

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Our design goal – complete humanization of mouse Hprt1

Today:

(March 23)

Next week:

(March 30)

April 13:TBD

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What’s NOT covered

We are designing a relatively simple engineering project

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We will (probably) not discuss:

  • Assembly of DNAs from multiple segments using yeast
  • Editing of DNAs in yeast (CREEPY)
  • Allele-specific engineering and verification
  • Methods of DNA delivery
  • Sequencing
  • Readout/phenotyping

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Resources – Our shared Google Doc

We (that is 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.)

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Recordings will be shared after each session

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Prerequisites

  • Read (or at least skim through) recommended papers
  • Install SnapGene and familiarize yourself with basic functionalities
  • Basic knowledge of:
    • CRISPR/Cas9
    • Golden Gate Assembly
    • PCR
    • BLAT/BLAST

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Introductions

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Our design goal – complete humanization of mouse Hprt1

Today:

(March 23)

Next week:

(March 30)

April 13:TBD

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Our design goal – complete humanization of mouse Hprt1

Today’s tasks:

  1. Identify the locus boundaries of mouse Hprt1 to be overwritten
  2. Identify gRNA sequences to cut at those boundaries
  3. Design pSpCas9 expression plasmids expressing these gRNAs
  4. Design a landing pad plasmid with homology arms targeting the mHprt1 locus
  5. Design a PCR genotyping strategy to identify candidate clones

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Locus boundaries - Considerations

Read more here: https://www.thedarkmatterproject.org/getting-started

  • Biology:
    • Genes, promoters, enhancers, CTCF sites, neighbors…
  • Mappability
    • Proxy for sequence repetitiveness
  • GC content
    • Avoid extremes
  • Availability of good gRNA sites
    • (and genotyping primers)
  • Type IIs RE sites
    • Can hinder landing pad plasmid Golden Gate assembly
  • Overall size
  • Assembly method

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Golden Gate Assembly

https://www.snapgene.com/guides/golden-gate-assembly