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.
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:
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:
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.
Note: It is recommended to keep each of the clones in culture while conducting validation studies as a back-up.
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 | $ | $$ or $$$ | $$$$ |
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. |
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:
>>> abm sells a Mismatch Cleavage Detection Kit for quick and easy CRISPR screening.
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.
>>> Try abm's SpeedySeq DNA Sequencing Service for fast, inexpensive CRISPR screening.
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:
>>> Try abm offers a wide range of NGS services for CRISPR screening and validation, including Amplicon Sequencing for CRISPR validation and biased off-target evaluation, and Whole Genome Sequencing for unbiased off-target evaluations
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.
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