Although the CRISPR system for genome editing is efficient, not every cell in a population will be edited. Therefore, it is important to understand how to screen and validate edited cells efficiently.
Before starting your experiment, it’s important to understand the possible outcomes of a CRISPR edit. Once sgRNA(s) 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:
Edited cells may be screened differently depending on whether the edit was repaired using Homology Directed Repair (HDR), or Non-Homologous End Joining (NHEJ). The HDR pathway is used for specific edits by including a HDR template along with sgRNA(s) and Cas9. The sequence of the HDR template is used to repair the cut site, integrating the desired sequence at the cut region. In comparison, NHEJ is error-prone and causes indel mutations (short insertions or deletions) at the cut site.
Here are some possible ways to screen for cells edited with an HDR template:
HDR events tend to be less common than indels created by NHEJ, so plan to screen a large number of colonies unless an enrichment method like FACS or antibiotic resistance is used.
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We offer Custom CRISPR HDR Templates for all your gene knock-in needs. |
Indels generated by NHEJ can be identified using a mismatch cleavage assay (commonly known as Surveyor), sequencing, or High Resolution Melting Analysis (HRMA) methods.
Table 1 — Comparison of Indel Screening Methods.
Mismatch Cleavage Detection Assay | Sanger Sequencing | Next Generation Sequencing | High Resolution Melting | |
Sensitivity (detection limit of mutant DNA) | 0.5-3% | 1-2% | 0.01% | 2% |
Mutation Sequence? | No | Yes | Yes | No |
Cost per Assaya | $ | $$$$$ | $$$$ | $ |
Mixed population screening? | Yes | No | Yes | No |
Clonal cell line screening? | Yes, spiked with WT DNA | Yes | Yes | Yes |
Distinguishes heterozygosity from homozygosity? | No | Yes | Yes | Yes |
High throughput? | Yes | No | Yes | Yes |
Advantages | Simple, fast. | Simple, gives sequence information. | Very sensitive, gives sequence information. | Fast, non-destructive, distinguishes heterozygosity/ homozygosity. |
Disadvantages | Polymorphic locus will lead to false positives. | May not distinguish heterozygous editing events if there is a high copy number. | Expensive, cannot detect large indels. | Set up cost for machinery can be high, cannot detect large indels. |
a. Estimated cost per assay. $: < 1 USD; $$: < 5 USD, $$$: > 100 USD; $$$$: > 500 USD.
Source: “Detection of on-target and off-target mutations generated by CRISPR/Cas9 and other sequence-specific nucleases” Zischewski et al. Biotechnology Advances, Feb 2017, Vol. 35, pp. 95-104.
The 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.
General protocol for the mismatch cleavage assay:
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Our Genomic Cleavage Detection Kit uses this method! |
Sanger Sequencing
Sanger sequencing is mainly used to investigate individual clonal cell lines. The typical method is to amplify the targeted 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.
An alternate method can be used. By sequencing PCR amplicons directly, one can save on time and labour. However, potential problems arise when dealing with non-homozygous editing events. Alleles with a different sequence will generate a chromatogram with multiple traces and superimposed peaks. This may be simple enough when dealing with only two sequences, but in the case of polyploid organisms or copy number variations, it can result in many overlapping traces which are difficult to distinguish. Decoding these overlapping traces can be aided by using a program like DSDecode (5), TIDE (2), or CRISP-ID (6).
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Did you know that abm offers Sanger Sequencing? Our Amplicon DNA Sequencing Service is a great choice for CRISPR screening. |
Next Generation Sequencing
Another popular method for detecting indels is Next Generation Sequencing. This can be done either on a mixed population, or on clonal cell lines which are pooled for a high-throughput approach to screening.
For performing high throughput NGS screening:
NGS methods have several advantages over other screening methods. They are able to detect whether all alleles of a gene were correctly edited, exactly what indels were generated, and whether a cell population is truly monoclonal. However, these methods are more costly and are more complicated to analyse.
NGS sequencing is also an excellent method for investigating off-target effects of CRISPR editing (see our upcoming article: CRISPR Cas9 – Evaluating Off-Target Effects).
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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 evalutions. |
Next Generation Sequencing
This method involves the analysis of the melt curve generated when performing real-time PCR in the presence of an intercalating fluorescent dye (similarly to qPCR). When the DNA is annealed in a double stranded form, the dye fluoresces. As the DNA is exposed to progressively higher temperatures, the dye is released and loses its fluorescent properties. By gathering this information, a melt curve is generated showing the temperature-dependent denaturation profile of the amplicons.
Each type of genome edit (wild-type, heterozygous mutation, biallelic mutation, or homozygous mutation) will generate a different melt curve. Therefore, HRMA makes it possible to distinguish between different mutant alleles. As well, the process leaves the amplicons intact, so they can be easily sequenced to determine the exact sequence of the edited region. Set up costs can be high, as HRMA machines cost from $10-25k. However, this is potentially mitigated by combining an existing qPCR machine with free HRMA software such as uAnalyze (8).
Other Indel Screening Methods
While we’ve covered some of the most popular screening methods above, there are many more that have been developed. Here is a brief summary of some other methods for CRISPR indel screening.
For more information about our experiences with CRISPR screening, see our CRISPR Cas9 Case Studies.