1st BASE CRISPR Screening Methods and Their Comparison

Want to speed up your CRISPR experiments?HRMT7E1 AssaySanger Sequencing

NGSFLA-PCR
 

 

 

 

T7E1 Mismatch Cleavage Assay

Mismatch cleavage assays such as the T7E1 mismatch cleavage assays have been widely adopted due to their relative simplicity, speed and cost-effectiveness. They can be used on single clones or pooled samples. They are typically used to preliminarily screen and identify clones for more detailed analysis such as by sequencing.

These assays rely on pairing or hybridisation between the edited and wild-type strands of the host DNA. The nuclease detects mismatches on the hybridised strands and cleave them. The resulting fragments are dissimilar in size and can be visualised using conventional gel electrophoresis.

Limitations:

One limitation of these assays is the lack of sequence-level information. In addition, the T7E1 assay misses SNPs and small INDELs (Vouillot et al., 2015). Furthermore, the assay may require optimisation of various experimental parameters due to its sensitivity to reaction conditions. One should note that to detect homozygous mutations, wild-type DNA has to be added to the PCR step for the formation of hetero-duplexes.

Mismatch Assay Mutation Screening Service includes:

  • QC of gDNA from targeted and control cells
  • Design and synthesis of PCR primers to amplify wild-type cell gDNA and edited type cell gDNA
  • PCR optimization, PCR set up and Clean up
  • T7E1 digestion for Heteroduplexes formation
  • Heteroduplexes digestion and QC
  • Deliverables: Gel electrophoresis Analysis Report

 

 

 

 

HRM (High-Resolution Melting)

High Resolution Melting (HRM) analysis is a post-PCR analysis method which enables identification of variations in nucleic acid sequences. HRM involves analysis of melting curves generated from performing real-time PCR in the presence of a dsDNA binding fluorescent dye. During melting, the dye is released and emits fluorescence as the dsDNA denature. The fluorescent intensity is plotted against the melting temperature to give the raw melt curve data. Each type of genome edits (wild-type, heterozygous mutation, bi-allelic mutation, or homozygous mutation) will generate a distinct amplicon-specific melting curve. By analysing the melting curve, it is possible to distinguish between different mutant alleles. The sensitivity and accuracy of the HRM-based method enables detection of CRISPR/Cas9-induced mutations from as early as a 2-cell stage zebrafish embryo (Samurat et al., 2016). HRM shows good detection reliability even for single-base pair INDEL; results show that less than 5% of mutated DNA containing one basepair INDEL can be detected using HRM (Denbow et al., 2017).

HRM analysis requires a simple set-up and involves minimal pipetting steps compared to enzymatic or SDS-PAGE methods. Then entire flow from genomic DNA extraction to PCR and finally HRM analysis can be completed in as little as 5 hours. This enables rapid high throughput screening of mutations.

Limitations:

One disadvantage of HRM analysis is the requirement of a dedicated software. An alternative is the melting temperature analysis function in a regular real-time-PCR machine. However, it has been shown that melt curve analysis in regular real-time-PCR has decreased sensitivity and takes a longer time than HRM (Denbow et al., 2017).

HRM qPCR Mutation Screening Service includes:

  • QC of gDNA
  • Design and synthesis of PCR primers to amplify region containing target site
  • PCR with reagent containing HRM fluorescent dye
  • Melt curve generation
  • Deliverables: HRM amplification curve, Normalized melt graph, Different graph, and HRM melt analysis & HRM analysis report

 

 

 

 

 

Sanger Sequencing

The gold standard method to identify induced mutations at the target locus entails amplifying the target region by PCR, then cloning the amplicon into a vector, followed by Sanger sequencing of the amplicon directly. Each vector should carry only one gene copy, which generates a clean trace in the chromatogram after sequencing. This method is preferably used to investigate individual clonal cell lines. The advantage of sequencing methods is the information on the type and frequency of mutations at the target locus.

Limitations:

In order to capture all gene copies, many colonies have to be sequenced. This process is laborious and time-consuming. Moreover, while direct Sanger sequencing of mixed populations/pooled clones is also possible, it is not recommended because the resulting chromatogram contains multiple overlapping traces that are difficult to differentiate (Bell et al. 2014). This is also observed for polyploid or diploid organisms with heterozygous or bi-allelic mutations. Overlapping traces can be automatically decoded by bioinformatics tools such as DSDecode or TIDE. The latter is able to detect INDELs with a sensitivity of 1-2% across various target regions in a pool of cells (Brinkman et al., 2014).

Sanger Sequencing Screening Service includes:

  • QC of Plasmid/PCR amplicon
  • BigDye® Terminator v3.1 Cycle Sequencing Reaction set up and Post Sequencing clean up
  • ABI 3730XL Sequencer run
  • Deliverables: Electropherogram (.ab1 format) and sequences (.seq fasta format)

 

 

 

 

NGS(Next Generation Sequencing)

Next Generation Sequencing (NGS) enables INDEL detection in both mixed populations and clonal cell lines. Its ability to process a large number of samples (samples are pooled together for a high-throughput sequencing run) and simultaneously screen off-target changes makes it a popular choice among researchers.

The data obtained from targeted amplicon sequencing is highly sensitive with detection levels as low as 0.01% (Hendel et al. 2015). This means that the researcher can be relatively certain that their samples do not contain off-target mutations if they are undetectable using this technique.

Besides being able to detect if all alleles of a gene were correctly edited, information on INDEL location and whether a cell population is truly monoclonal can be derived from NGS data.

Limitations:

The NGS method is cost-effective only for larger sample numbers. Furthermore, NGS data typically requires analysis by a bioinformatician. Also, because of its relatively short reads, the method misses larger INDELs.

Next Generation Sequencing Mutation Screening Service includes:

  • QC of gDNA
  • Design and synthesis of PCR primers to amplify region containing target site
  • Library preparation and QC
  • MiSeq 150/250/300 PE reads run
  • Deliverables: Raw Data and after run QC Analysis Report

 

 

 

 

FLA-PCR(Fragment Analysis)

Fragment analysis is a capillary electrophoresis (CE) based method for AFLP, MLPA & SNP detection. CE is a proven sensitive, high throughput and high-resolution system for nucleic acid analysis.

Recently multiple fragment analysis methods such as IDAA, Fluorescent PCR and CRISPR-STAT have been developed in CRISPR/Cas9 genome editing studies. These are reported to be fast, sensitive, precise and cost-effective methods for mutation detection. Moreover, CE instrumentation with Sanger sequencing capability, e.g. Thermo Fisher 3130 and 3730 series, enables single base resolution.

Fragment analysis assays have a simple two-step protocol (PCR followed by CE) independent of enzymatic cleavage. It has been reported that their sensitivity and resolution are comparable to NGS with an INDEL detection sensitivity of about 0.1% (Lonowski et al., 2017). Hence they can be used in both basic research and more challenging genome editing applications such as therapeutic INDEL profiling.

Fragment analysis assays are promising for various applications. It has been reported by Gardner et al. (2016) as “an analytical tool [which] fundamentally changes the scale and complexity of experimental design”. According to them, high-throughput CE assays based on fluorescently labelled oligonucleotides can be designed to characterise, discover, engineer, or screen nucleic acid metabolic enzymes. Such studies include that on DNA repair, recombination, restriction, modification and RNA metabolism. Furthermore, it has been applied as reagent quality control assays.

Limitations:

The bottleneck for fragment analysis methods lie in secondary data analysis. While there is no limiting factor for primary data processing (baselining, peak detection and sizing), current software such as Gene Mapper and Peak Scanner (both from Thermo Fisher) do not fully satisfy data analysis requirements for new applications such as fragment labelling and quantification, and particularly targeting efficiency calculations for genome editing studies.

FLA-PCR Mutation Screening Service includes:

  • QC of gDNA
  • Design and synthesis of PCR primers to amplify wild-type cell gDNA and edited type cell gDNA
  • PCR optimization, PCR set up and Clean up
  • FA RUN set up using LIZ dye marker
  • Deliverables: Efficiency of edits Analysis Report

 

Review Article

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