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Screening and Validation of Knockout


 
 

Welcome to our training series on performing your own CRISPR Cas9 experiment for gene knockout. This is our last session. This week, we will help you decide how to screen and validate your cell line, and describe various methods in detail.

 
 

This Week We Will Learn:


 
 

CRISPR-Crash-Course-check-icon_v2-08

Screening Basics

 

CRISPR-Crash-Course-check-icon_v2-08Protocol for Monoclonal Selection

 

CRISPR-Crash-Course-check-icon_v2-08Comparison of Indel Screening Methods

Cas9-character-holding-a-book-07
 
 

1. Screening Basics


 
 

First, it’s important to understand the possible outcomes of a CRISPR edit. Once sgRNA and Cas9 have been introduced to a cell line, the generated edits will not be identical, nor will they necessarily occur in all alleles of a gene or all cells in a population. For example, when targeting a single gene of a diploid cell there are four possible outcomes:

  1. No edit occurs,
  2. One allele is edited (heterozygous mutation),
  3. Both alleles are edited, but they carry a different sequence (biallelic mutation),
  4. Both alleles are edited, and they carry the same sequence (homozygous mutation).

 

In most cases, a biallelic or homozygous mutation would be desired in order to be sure the gene of interest is completely edited and none of the wild type phenotype remains.

 

The basic workflow of screening for edited cells is:

  • Perform your first screening assay on a mixed population to determine whether a significant number of cells developed the desired genome edit. The most common method for polyclonal screening is the Mismatch Cleavage Detection Assay (a.k.a. the Surveyor or T7E1 Assay).
  • If some of the population was successfully edited, isolate single cells. This is usually done using serial dilution.
  • Expand isolated cells to create clonal cell lines.
  • Perform screening on each clonal cell line, until one is found with the desired edit.

 

CRISPR-Repair-Mechanisms

Summary of two dsDNA cleavage repair pathways: NHEJ (Non-Homologous End Joining) and HDR (Homology Directed Repair).
 
 

2. Protocol for Monoclonal Selection


 
 

Proceed with monoclonal selection after determining that some of the population has been edited. This protocol describes how to isolate single cells from a clonal pool using two dilution series.

  1. First, add 100 μl of culture media (containing the selection drug at the concentration determined in the killing curve) to each well of the 96-well plate.
  2. Add 200 μl of initial cell suspension into well A1 (Figure 5).
  3. Proceed with the first dilution series (refer to Figure 5, red arrow). Make a 1:2 dilution series vertically by adding 100 μl from well A1 to B1. Mix well and then add 100 μl from well B1 to C1. Repeat until the last well (H1) is reached. Remove 100 μl from the last well to keep the total volume the same (100 μl) for all wells.
  4. Add 100 μl of media to each well of column 1 to make the volume 200 μl in total for each well.
  5. Proceed with the second dilution series (refer to Figure 5, blue arrows). Using a multichannel pipette, transfer 100 μl of cell suspension from column 1 to column 2 and so forth until column 12 is reached. Again, remove 100 μl from each well to keep the total volume consistent (100 μl) in each well.Monoclonal-Selection
  6. Add 100 μl of fresh media to each well in this plate so that the total volume in each well is 200 μl. Incubate the plate at 37°C, 5% CO2.
  7. It may require approximately 2 weeks for clones to appear. Allow individual monoclones to grow to form a monolayer and transfer cells to a 24-well plate followed by a 6-well expansion. The genomic DNA isolated from each of the resulting expanded clones can be used for further gene editing validation.

 

Note: It is recommended to keep each of the clones in culture while conducting validation studies as a back-up.

 
 

3. Comparison of Indel Screening Methods


 
 

NHEJ is error-prone and causes indel mutations (short insertions or deletions) at the cut site. See below for a comparison of different popular indel screening methods. Following this summary are more detailed descriptions of each.

 


Mismatch Cleavage Detection Assay

Sanger Sequencing

Next Generation Sequencing

Sensitivity (detection limit of mutant DNA)

0.5-3%

1-2%

0.01%

Mutation Sequence?

No

Yes

Yes

Cost per Assaya

$

$$/$$$

$$$$

Mixed population screening?

Yes

No

Yes

Clonal cell line screening?

Yes, spiked with WT DNA

Yes

Yes

Distinguishes heterozygosity from homozygosity?

No

Yes

Yes

High throughput?

Yes

No

Yes

Advantages

Simple, fast.

Simple, gives sequence information.

Very sensitive, gives sequence information.

Disadvantages

Polymorphic locus will lead to false positives.

May not distinguish heterozygous editing events if there is a high copy number.

Expensive, cannot detect large indels.


a. Mismatch Cleavage Detection Assay

The most widely used method to detect indels caused by CRISPR gene editing is the mismatch cleavage assay (a.k.a. the Surveyor assay). This assay relies on the Surveyor nuclease, which causes a double stranded break at the 3’ end of any mismatches between two strands of annealed DNA. This assay is sensitive to mutations that occur as low as 1 in 32 copies and is able to detect mismatches caused by up to 12 nucleotide indels.

 

Steps in the mismatch cleavage assay:

  • PCR amplify the edited region from a cell population.
  • Denature the strands, then re-anneal. This allows the strands of DNA to separate then randomly re-hybridize, creating heteroduplexes.
  • Treat the DNA with the T7 endonuclease. The endonuclease will cut only if the strands have formed a heteroduplex.
  • Run digested DNA fragments on an agarose gel. Cleaved product shows the presence of heteroduplexes, indicating indel formation.

 

CRISPR-Genomic-Cleavage-Detection-Kit-Surveyor-Assay

 

Workflow for the Mismatch Cleavage Detection Assay.

 

>>>  abm sells a Mismatch Cleavage Detection Kit for quick and easy CRISPR screening. 

 

b. Sanger Sequencing

Sanger sequencing is mainly used to investigate individual monoclonal cell lines. This is because polyclonal pools will include cells with many different variations at the target site: some will remain unedited, and others will have a variety of different indels. This can result in many overlapping traces which are difficult to analyze.

The typical method for screening of a monoclonal cell line is to amplify the target region by PCR then clone the amplicons into a vector. This way, each vector will carry only one gene copy, which generates a clean trace when sequenced. In order to determine the sequence of all gene copies, many colonies need to be screened. Although this method is considered the gold standard for indel detection, it can be expensive and time-consuming.

To analyse Sanger sequencing data, align the sample sequences with the sequence of the wildtype and the sgRNA. Insertions or deletions at the sgRNA target site that cause a frameshift mutation resulting in an early stop codon that will knockout the gene. To see if mutations cause an early stop codon, translate the mutated sequence using a translation tool such as ExPasy. For monoclonal bi-allelic knockout, all clones sequenced should show one of two indel sequence variations. If more than two sequences are obtained from one sample, it means that it’s likely your cell line is not actually monoclonal.

Sanger-Sequence-CRISPR-Validation

In this image, there are two frameshift mutations (4 nt and 2 nt deletions) compared to the reference and sgRNA target sequences (“C26 target direct” and “CCT sgRNA”).

 

>>>  Try abm's SpeedySeq DNA Sequencing Service for fast, inexpensive CRISPR screening.

 

b. Next Generation Sequencing

With Next Generation Sequencing hundreds of thousands of alleles can be sequenced at once, resulting in a more robust dataset. By contrast, Sanger sequencing is only feasible for 1-100 clones and therefore it can miss a large proportion of the population.

NGS screening can be done either on a mixed population, or on clonal cell lines which may be pooled for a high-throughput approach to screening.

For performing high throughput NGS screening:

  • Culture clonal cell lines in a 96 well plate. Establish a duplicate plate.
  • PCR amplify the edited region. Use barcoded primers to ensure that sequencing reads can later be matched with their original clone.
  • Pool the PCR amplified DNA.
  • Prepare and sequence the library.
  • Analyse sequencing data. This may be done using tools such as CRISPResso or CRISPR Genome Analyzer.
  • Identify clones with the desired mutation. Expand these clones from the duplicate plate as desired.

 

A) Before knockout, only WT sequences are detected.

B) After Cas9 and sgRNA delivery, the first round of selection shows a mixed distribution of indel and WT sequences.

C) After the second round of selection, only indel sequences remain.

 

>>>  abm offers a wide range of NGS services for CRISPR screening and validation, including Amplicon Sequencing for CRISPR validation and biased off-target evaluation, Whole Genome Sequencing for unbiased off-target evalutions, and sgRNA Deconvolution Screening for studying the effects of pooled gRNA libraries.

 

Thanks for joining us on this exciting journey from experimental design all the way to validation. We hope you’ve enjoyed this training course. If you have any questions or feedback, please feel free to contact us through the form below.

 
 

Test Your Knowledge!


 
 

Take our Week 4 Quiz, and graduate to becoming a Genome Editing Genius!

 

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