Cas9 nickase validation by Sanger sequencing analysis workflow: CaSanger.
CRISPR Cas9 is a RNA guided endonuclease now commonly utilized in a broad range of molecular biology applications. The native Cas9-sgRNA complex produces DNA double strand breaks when successfully targeted. Variants of Cas9 have been engineered with only one active catalytic site; resulting in th enzyme producing single strand DNA breaks (nicks) when successfully targeted. These Cas9 "nickases" are commonly used with 2 sets of sgRNAs which together produce double strand breaks.
A key component of any Cas9 assay is validation of both the presence and location of a specific break. There are a variety of methods to validate the presence of double strand DNA breaks both in vivo and in vitro but validation of a nick is more difficult largely due to difficulty of designing a PCR reaction that will produce target, and nick dependent products.
An alternative approach is to utilize Sanger sequencing which is sensitive to DNA perturbations and relies on linear amplification (utilizing a single primer). If a nicked DNA template is subjected to Sanger sequencing with a primer that binds to the same strand that is targeted by a nicking enzyme there should be no distinguishable signal beyond the nick. This is because if a sufficiently high percentage of the template has the same targeted nick, the polymerase used in the sequencing reaction will fall off the single stranded template at the site of nick.
The workflow requires conda and snakemake to run. See the
Snakemake documentation
for help getting these programs installing on your machine.
This is the main configuration file you need to worry about. The workflow uses the parameters and filepaths specified here to function properly. Each row of this file corresponds to one "validation" experiment, i.e. you want to show a specific sgRNA successfully targets your nickase to the target site and cleaves.
| Field | Description |
|---|---|
| sgRNA_fullname | Long form name of the sgRNA you are validating; must be unique. |
| sgRNA_prefered_name | Short form of sgRNA you are validating; must be unique. |
| target_PAM | Target site of sgRNA from 5' -> 3' with the PAM site. |
| cas9-sgRNA-read | Path to abi (trace file) from read treated with Cas9 and sgRNA. |
| cas9-read | Path to abi (trace file) from read treated with only sgRNA. |
| template-read | Path to abi (trace file) from read with no treatment (template only). |
| template_name | Name of template DNA; must be unique. |
| template_gb | Path to genbank file of template DNA sequence. |
| primer_name | Name of primer used in the sequencing reactions. |
| primer_seq | Sequence of primer in the 5' -> 3' direction. |
| read_date | Date of Sanger reactions. (Delim with - to avoid possible file path parsing issues). |
Main output produced are plots showing read alignments and traces for each sgRNA. An example plot produced by the workflow is shown below.
Plots show the Sanger read traces of each
tested condition and highlight the region
of the trace where the nick is expected to
terminate the read. The bottom most subplot
is a map of the template zoomed in over the
entire region of the longest aligned read. The workflow
will annotate the locations of all read alignments,
the sgRNA target site, and the primer used for sequencing along with any other features already
present in the supplied template genbank file.
Both png and pdf versions of the plot are produced and will be located in
output/{run_name}/pots/{sgRNA}/ directory where {} indicate variables (snakemake wildcards).
- Currently the plotting script does not taken into account gaps in alignment of the Sanger read to the DNA template. This can result in a disparity of a few bases when viewing alignments.