At the Genome Engineering Core (GECO), our aim is to develop genome engineering tools that unravel the complex genetic underpinnings of diseases. We use these tools in collaboration with researchers working to understand and treat a broad range of human diseases, driving medical discovery and the translation of genetic insights into therapeutic solutions. 

GECO helps collaborators in experimental design and execution by helping them choose the best model system, perturbation type, challenge, and readout for their goals. Our tools include CRISPR knockout, interference, base editing, CRISPRoff, and ORF overexpression. Typical readouts include amplicon sequencing and scRNA-seq. 

Our technologies are divided into the categories of genome engineering and spatial profiling. For descriptions, see below. For pricing or questions, go to iLab or email us at: genome@columbia.edu 

Genome Engineering

Genome engineering is the targeted modification of the genetic material of cells. Examples include targeted insertions, deletions, edits, or epigenetic modifications to the genome. The purpose can be either research, such as understanding the cause of a disease, or therapeutic, as in the case of treating a disease. Although GECO is proficient in multiple technologies, currently the most used is CRISPR.

1. To create a genome engineering project:
2. Schedule a planning meeting with GECO (optional for ready-to-screen libraries).
3. Order the library on iLab.
4. Schedule a pickup with us (bring a container of dry ice to pick up the virus).
5. Return gDNA.

CRISPR Technologies

CRISPR experiments have four dimensions1:  

1. The Model: cell lines, primary cells, organoids, or in vivo.
2. The Perturbation: CRISPR KO, CRISPRi, CRISPRa, base editing, etc.
3. The Challenge: Proliferation, drug resistance, pathogen exposure, etc.
4. The Readout: sgRNA or amplicon sequencing, single-cell RNA sequencing, multi-omics, or imaging.  

GECO can work with you on several dimensions of your experiment, including choosing the optimal perturbation and readout. We have ready-to-screen, validated lentiviral sgRNA libraries. We may also be able to make a custom library for your experiment. 

Perturbations:

1.  CRISPR KO: The classic CRISPR experiment involves targeting a Cas endonuclease to a gene of interest and creating a double-strand break in the DNA. The repair of the break can result in a nonsense mutation or other event that destroys the gene’s function. 
   1. Pros: 
      1. Well established technology that generally has good efficiency. 
      2. Does not require long-term maintenance of Cas protein.
   2. Cons:
      1. Having too many double-strand breaks in the genome at the same time can induce DNA damage response. This is more likely to occur in high-ploidy cell lines, or when cutting an amplified gene.  Induction of the DNA damage response can lead to an unintended selection of cells with misregulation of p53 pathway. 
      2. Transient off-target events may have permanent consequences (compare to CRISPRi)
      3. Study of early transcriptional dynamics may be difficult since detection and repair of double-strand breaks takes time, and may occur at different times in different alleles or in different cells. 
2. CRISPRi: A catalytically dead Cas protein (dCas9 or dCas12a), typically fused to one or more repressor domains, is targeted to a promoter or enhancer, where it creates a steric block. In the case of a promoter, the transcriptional machinery cannot assemble or proceed, and transcription is transiently inhibited (lasting as long as the block is maintained).
   1. Pros:
      1. No double-strand breaks. 
      2. Potentially low off-target effects since 1) repression is unlikely to occur unless dCas9 binds within a small (~250nt) window near the TSS, and 2) dCas9 must remain bound to the site for significant repression to occur.
      3. Transcriptional repression can occur as soon as sufficient dCas9 is present. In an inducible system, this could be as ~6-18 hours.   
   2. Cons:
      1. Repression may be impossible or inadequate for some genes. There is variation in knockdown efficacy, and some genes may not be knocked down significantly for various reasons.
      2. dCas9 must be maintained in order to maintain repression. This can cause problems in some contexts. 
3. CRISPRoff: A catalytically dead Cas protein (dCas9), fused to both repressor and methylase domains, is targeted to a gene promoter. Genes that contain CpG islands in their promoters can be durably repressed—i.e., epigenetic repression that persists in daughter cells. 
   1. Pros:
      1. Hereditable epigenetic silencing may be achieved for many genes.
      2. May be used to repress gene expression in contexts that would not be amenable to other techniques.
   2. Cons:
      1. The CRISPRoff fusion protein is large. 
      2. Limited to genes that can be silenced by methylation of their promoters.
4. CRISPR base editing: A nickase Cas protein (nCas9 or nCas12a) is fused to a deaminase domain and targeted to a site where it causes either C/T, A/G, or C/G conversions without inducing a double-strand break. Base editing is often used to perform targeted saturation mutagenesis (e.g., to identify drug resistance mutations) or to correct alleles that cause disease.
   1. Pros:
      1. The ability to create precise edits in genes without creating double-strand breaks.
      2. Powerful tool for identifying drug resistance mutations and drug-target binding points.
      3. Good efficacy and product purity.
   2. Cons:
      1. Cannot make any possible edit. That is, it’s not possible to substitute any amino acid at the residue of interest.
      2. Some editors are toxic in some cell lines.
5. CRISPRa: A catalytically dead Cas protein (dCas9 or dCas12a), typically fused to one or more activator domains, is targeted to a promoter, where it recruits transcriptional machinery. 
   1. Pros:
      1. Activation of endogenous gene, including context-specific isoforms from endogenous promoter.
   2. Cons:
      1. Activation may not be possible for many genes.

Other Genome Engineering Technologies:Extend

1. ORF overexpression: The coding sequence of a gene is ectopically expressed
   1. Pros:
      1. Established technology.
      2. Strong overexpression of most ORFs.
   2. Cons:
      1. Exogenous overexpression.
      2. Often fails for very large ORFs or GC-rich ORFs. 

Proliferation/survival screens

In a typical proliferation screen, the model (e.g., cell line) is first transduced with enAsCas12a and then with the sgRNA library. Genome-wide sgRNA libraries usually contain 20,000 – 60,000 constructs. At all points in the screen, it is important to maintain adequate coverage of the library. Often, this means 1,000X coverage, but in some cases lower coverage is sufficient. Consequently, the number of cells required for a screen can become quite large. For example: 60,000 (sgRNA) * 1000 (coverage) * 3 (replicates) * 3 (conditions) * 2 (timepoints) =  1*109 cells. Since the sgRNA library must be transduced at a low %infected to prevent multiple guides in the same cell, for the screen in this example, 4 billion cells would need to be infected.

Proliferation screens often last for ≥10 cell divisions, or 14-21 days, after which gDNA is isolated. Because an entire genome must be isolated for each ~350nt amplicon, in the example above 2,376 micrograms of gDNA would have to be used as input for the amplicon sequencing PCR. Since PCR reactions use 1-10ug of gDNA input per reaction, many PCR reactions must be performed and validated prior to sequencing. This is best done using a liquid-handling robot.

We generally recommend enAsCas12a with a multi-sgRNA construct for proliferation screens. AsCas12a, a CRISPR-associated protein derived from Acidaminococcus sp., is a common component in CRISPR genome-editing systems. Compared to Cas9 it offers several advantages, including smaller size and the ability to process its own CRISPR RNA (guide). These make it well-suited for multigenic experiments and for targeting multiple locations on a single gene. enAsCas12a has been engineered to have a less restrictive PAM2, which increases the number of potential target sites and improves performance in CRISPR KO screens.3

CRISPR-based screens with scRNA-seq readout (Perturb-seq)

CROP-seq4, Perturb-seq5,6, and CRISP-seq7 are technologies that combine CRISPR-mediated perturbations with single-cell RNA sequencing readout. Newer iterations of these technologies use direct capture of the sgRNA as a means of detecting which perturbation(s) occurred in each cell. They are powerful tools that, at the extreme, allow for up to genome-wide RNA-seq profiling in a single experiment. Often, these screens are short, with scRNA-seq occurring 1-8 days after the constructs are virally transduced. 

Other types of Screens

GECO can help with other types of screens, including FACS screens and ORF overexpression screens. 

Spatial Profiling

High-throughput in situ spatial profiling enables the mapping of RNA or protein expression within individual cells in their native spatial context within a tissue. Maintaining spatial orientation allows researchers to understand how cellular function is influenced by location within a tissue.

To facilitate high-throughput spatial profiling projects, GECO has a 10X Genomics Xenium. The Xenium performs spatial profiling of hundreds or thousands of RNAs and multiplexed protein in cells and tissues with subcellular resolution.

To use the Xenium: 

1. Schedule a Xenium run in iLab and pick up a Xenium slide.
2. Have your tissue(s) sectioned according to 10X Genomics’ guidelines for FFPE or fresh frozen samples. Following these instructions is essential to obtaining good data.
3. Schedule a drop-off with GECO.

1. Bock, C. et al. High-content CRISPR screening. Nat Rev Methods Primers 2 (2022). https://doi.org:10.1038/s43586-022-00098-7
2. Kleinstiver, B. P. et al. Engineered CRISPR–Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing. Nature Biotechnology 37, 276-282 (2019). https://doi.org:10.1038/s41587-018-0011-0
3. DeWeirdt, P. C. et al. Optimization of AsCas12a for combinatorial genetic screens in human cells. Nat Biotechnol 39, 94-104 (2021). https://doi.org:10.1038/s41587-020-0600-6
4. Datlinger, P. et al. Pooled CRISPR screening with single-cell transcriptome readout. Nat Methods 14, 297-301 (2017). https://doi.org:10.1038/nmeth.4177
5. Adamson, B. et al. A Multiplexed Single-Cell CRISPR Screening Platform Enables Systematic Dissection of the Unfolded Protein Response. Cell 167, 1867-1882 e1821 (2016). https://doi.org:10.1016/j.cell.2016.11.048
6. Dixit, A. et al. Perturb-Seq: Dissecting Molecular Circuits with Scalable Single-Cell RNA Profiling of Pooled Genetic Screens. Cell 167, 1853-1866 e1817 (2016). https://doi.org:10.1016/j.cell.2016.11.038
7. Jaitin, D. A. et al. Dissecting Immune Circuits by Linking CRISPR-Pooled Screens with Single-Cell RNA-Seq. Cell 167, 1883-1896.e1815 (2016). https://doi.org:10.1016/j.cell.2016.11.039