---
title: "Introduction to crisprViz"
author:
- name: Luke Hoberecht
affiliation: Data Science and Statistical Computing, gRED, Genentech
email: hoberecl@gene.com
date: "`r Sys.Date()`"
output:
BiocStyle::html_document:
toc_float: true
#theme: paper
number_sections: true
vignette: >
%\VignetteIndexEntry{Introduction to crisprViz}
%\VignetteEngine{knitr::rmarkdown}
\usepackage[utf8]{inputenc}
---
```{r, include=FALSE}
knitr::opts_chunk$set(message=FALSE, warning=FALSE)
```
# Introduction
The `crisprViz` package enables the graphical interpretation of `GuideSet`
objects from the [crisprDesign](https://github.com/crisprVerse/crisprDesign) package by plotting guide RNA (gRNA)
cutting locations against their target gene or other genomic region.
This vignette walks through several use cases that demonstrate the range of
and how to use plotting functions in the `crisprViz` package. This vignette
also uses our core gRNA design package [crisprDesign](https://github.com/crisprVerse/crisprDesign)to manipulate `GuideSet` objects in
conjunction with plotting in the process of gRNA design.
Visit our [crisprVerse tutorial page](https://github.com/crisprVerse/Tutorials) to learn more about how to design gRNAs for different applications.
# Installation and getting started
## Software requirements
### OS Requirements
This package is supported for macOS, Linux and Windows machines. Packages
were developed and tested on R version 4.2.
## Installation from Bioconductor
`crisprViz` can be installed from Bioconductor using the following
commands in a fresh R session:
```{r, eval=FALSE}
install.packages("BiocManager")
BiocManager::install("crisprViz")
```
# Use cases
All examples in this vignette will use human genome assembly `hg38` from
the `BSgenome.Hsapiens.UCSC.hg38` package and gene model coordinates from
Ensembl release 104. We begin by loading the necessary packages.
```{r, message=FALSE, warning=FALSE}
library(BSgenome.Hsapiens.UCSC.hg38)
library(crisprDesign)
library(crisprViz)
```
## Visualizing the best gRNAs for a given gene
Suppose we want to design the four best gRNAs using the SpCas9 CRISPR
nuclease to knockout the human KRAS gene. To have the greatest impact
on gene function we want to prioritize gRNAs that have greater isoform
coverage, and target closer to the 5' end of the CDS.
Let's load a precomputed `GuideSet` object containing all possible gRNAs
targeting the the CDS of KRAS, and a `GRangesList` object describing the
gene model for KRAS.
``` {r}
data("krasGuideSet", package="crisprViz")
data("krasGeneModel", package="crisprViz")
length(krasGuideSet) # number of candidate gRNAs
```
For how to design such gRNAs, see the `crisprDesign` package. Before
we plot all of our candidate gRNAs, let's first generate a simple plot
with a few gRNAs to familiarize ourselves with some plot components and options.
```{r}
plotGuideSet(krasGuideSet[1:4],
geneModel=krasGeneModel,
targetGene="KRAS")
```
There are a few things to note here.
* The ideogram track and genome axis track are at the top of our plot and
give us coordinate information.
* Our `targetGene` KRAS is plotted next, using coordinates from
the provided gene model `krasGeneModel`, followed by our spacer subset.
The name of each track is given on the left.
* The strand information for each track is included in
the label: `<-` for reverse strand and `->` for forward strand.
* While we can identify which exon each spacer targets (which may
be sufficient), the plot window is too large to provide further information.
* The plot only shows the 3' end of KRAS, rather than the entire gene.
This last point is important: the default plot window is set by the
spacers' ranges in the input `GuideSet` object. We can manually adjust
this window by using the `from`, `to`, `extend.left`, and `extend.right`
arguments. Here is the same plot adjusted to show the whole KRAS gene,
which also reveals an additional isoform that is not targeted by any
spacer in this example subset.
```{r}
from <- min(start(krasGeneModel$transcripts))
to <- max(end(krasGeneModel$transcripts))
plotGuideSet(krasGuideSet[1:4],
geneModel=krasGeneModel,
targetGene="KRAS",
from=from,
to=to,
extend.left=1000,
extend.right=1000)
```
As calculated above, there are a total of `r length(krasGuideSet)`
candidate gRNAs targeting the CDS of KRAS. Including all of them could
crowd the plot space, making it difficult to interpret. To alleviate
this we can hide the gRNA labels by setting the `showGuideLabels`
argument to `FALSE`.
``` {r fig.height=10}
plotGuideSet(krasGuideSet,
geneModel=krasGeneModel,
targetGene="KRAS",
showGuideLabels=FALSE,
from=from,
to=to,
extend.left=1000,
extend.right=1000)
```
At the gene level, the plot window is too large to discern details for
each spacer target. However, we can see five distinct clusters of
spacer target locations that cover the CDS of KRAS. The spacers in
the 5'-most cluster (on the reverse strand) target the only coding
region of the gene that is expressed by all isoforms, making it an
ideal target for our scenario.
We can see which gRNAs target this region by returning `showGuideLabels` to
its default value of `TRUE`, and by adjusting the plot window to focus on
our exon of interest.
```{r}
# new window range around target exon
targetExon <- queryTxObject(krasGeneModel,
featureType="cds",
queryColumn="exon_id",
queryValue="ENSE00000936617")
targetExon <- unique(targetExon)
from <- start(targetExon)
to <- end(targetExon)
plotGuideSet(krasGuideSet,
geneModel=krasGeneModel,
targetGene="KRAS",
from=from,
to=to,
extend.left=20,
extend.right=20)
```
At this resolution we can get a much better idea of spacer location and
orientation. In particular, the PAM sequence is visible as a narrow box
on the 3' side of our protospacer sequences. We can also distinctly see
which spacer targets overlap each other--it may be best to avoid pairing
such spacers in some applications lest they sterically interfere
with each other.
If we have many gRNA targets in a smaller window and are not concerned
with overlaps, we can configure the plot to only show the `pam_site`,
rather than the entire protospacer and PAM sequence, by setting `pamSiteOnly`
to `TRUE`.
```{r}
plotGuideSet(krasGuideSet,
geneModel=krasGeneModel,
targetGene="KRAS",
from=from,
to=to,
extend.left=20,
extend.right=20,
pamSiteOnly=TRUE)
```
Let's filter our `GuideSet` by the spacer names in the plot then pass an
on-target score column in our `GuideSet` to `onTargetScores` to color the
spacers according to that score, with darker blue colors indicating higher
scores. Note that for this plot we need not provide values for `from` and
`to`, as the plot window adjusts to our filtered `GuideSet`.
```{r, message=FALSE, warning=FALSE}
selectedGuides <- c("spacer_80", "spacer_84", "spacer_88", "spacer_92",
"spacer_96", "spacer_100", "spacer_104", "spacer_108",
"spacer_112")
candidateGuides <- krasGuideSet[selectedGuides]
plotGuideSet(candidateGuides,
geneModel=krasGeneModel,
targetGene="KRAS",
onTargetScore="score_deephf")
```
<!--  -->
## Plotting for precision targeting
For a given CRISPR application, the target region may consist of only
several base pairs rather than an exon or entire gene CDS.
In these instances it may be important to know exactly where the gRNAs
target, and plots of gRNAs must be at a resolution capable of
distinguishing individual bases. This is often the case for
CRISPR base editor (CRISPRbe) applications, as the
editing window for each gRNA is narrow and the results
are specific to each target sequence.
In this example, we will zoom in on a few gRNAs targeting the 5' end of
the human GPR21 gene. We want our plot to include genomic
sequence information so we will set the `bsgenome` argument
to the same `BSgenome` object we used to create our `GuideSet`.
First, we load the precomputed `GuideSet` and gene model objects for GPR21,
```{r}
data("gpr21GuideSet", package="crisprViz")
data("gpr21GeneModel", package="crisprViz")
```
and then plot the gRNAs.
```{r, fig.height=4, fig.width=6}
plotGuideSet(gpr21GuideSet,
geneModel=gpr21GeneModel,
targetGene="GPR21",
bsgenome=BSgenome.Hsapiens.UCSC.hg38,
margin=0.3)
```
The genomic sequence is given at the bottom of the plot as color-coded boxes.
The color scheme for describing the nucleotides is
given in the
[**biovizBase** package](https://bioconductor.org/packages/3.16/bioc/html/biovizBase.html).
If the plot has sufficient space, it will display nucleotide symbols rather
than boxes. We can accomplish this by plotting a narrower range
or by increasing the width of our plot space (see "Setting plot size" section).
The plot above was generated with a plot space width of 6 inches;
here's the same plot after we increase the width to 10 inches:
```{r, fig.height=4, fig.width=10}
# increase plot width from 6" to 10"
plotGuideSet(gpr21GuideSet,
geneModel=gpr21GeneModel,
targetGene="GPR21",
bsgenome=BSgenome.Hsapiens.UCSC.hg38,
margin=0.3)
```
## CRISPRa and adding genomic annotations
In this scenario we want to increase expression of the human MMP7 gene
via CRISPR activation (CRISPRa). We will use the SpCas9 CRISPR nuclease.
```{r}
data("mmp7GuideSet", package="crisprViz")
data("mmp7GeneModel", package="crisprViz")
```
The `GuideSet` contains candidate gRNAs in the 2kb window immediately upstream
of the TSS of MMP7. We will also use a `GRanges` object containing repeat
elements in this region:
```{r}
data("repeats", package="crisprViz")
```
Let's begin by plotting our `GuideSet`, and adding a track of repeat
elements using the `annotations` argument. Our `guideSet` also contains
SNP annotation, which we would also prefer our gRNAs to not overlap.
To include a SNP annotation track, we will set `includeSNPTrack=TRUE` (default).
```{r}
from <- min(start(mmp7GuideSet))
to <- max(end(mmp7GuideSet))
plotGuideSet(mmp7GuideSet,
geneModel=mmp7GeneModel,
targetGene="MMP7",
guideStacking="dense",
annotations=list(Repeats=repeats),
pamSiteOnly=TRUE,
from=from,
to=to,
extend.left=600,
extend.right=100,
includeSNPTrack=TRUE)
```
Some of our candidate gRNAs target repeat elements and likely target a
large number of loci in the genome, potentially causing unintended
effects, or overlap with SNPs, which can reduce its activity. Let's
remove these gRNAs and regenerate the plot.
```{r}
filteredGuideSet <- crisprDesign::removeRepeats(mmp7GuideSet,
gr.repeats=repeats)
filteredGuideSet <- filteredGuideSet[!filteredGuideSet$hasSNP]
plotGuideSet(filteredGuideSet,
geneModel=mmp7GeneModel,
targetGene="MMP7",
guideStacking="dense",
annotations=list(Repeats=repeats),
pamSiteOnly=TRUE,
from=from,
to=to,
extend.left=600,
extend.right=100,
includeSNPTrack=TRUE)
```
Note how removing gRNAs that overlap SNPs from our `GuideSet` also
removed the SNP track. To prevent plotting an empty track, `plotGuideSet`
will only include a SNPs track if at least one gRNA includes SNP
annotation (i.e. overlaps a SNP).
Conversely, there are specific genomic regions that would be beneficial to
target, such as CAGE peaks and DNase I Hypersensitivity tracks. We show in
the `inst\scripts` folder how to obtain such data from the Bioconductor
package `AnnotationHub`, but for the sake of time, we have precomputed those
objects and they can be loaded from the `crisprViz` package directly:
```{r}
data("cage", package="crisprViz")
data("dnase", package="crisprViz")
```
We now plot gRNAs alongside with those two tracks:
```{r}
plotGuideSet(filteredGuideSet,
geneModel=mmp7GeneModel,
targetGene="MMP7",
guideStacking="dense",
annotations=list(CAGE=cage, DNase=dnase),
pamSiteOnly=TRUE,
from=from,
to=to,
extend.left=600,
extend.right=100)
```
Let's filter our `GuideSet` for guides overlapping the
plotted DNase site then regenerate the plot.
```{r}
# filter GuideSet for gRNAs overlapping DNase track
overlaps <- findOverlaps(filteredGuideSet, dnase, ignore.strand=TRUE)
finalGuideSet <- filteredGuideSet[queryHits(overlaps)]
plotGuideSet(finalGuideSet,
geneModel=mmp7GeneModel,
targetGene="MMP7",
guideStacking="dense",
annotations=list(CAGE=cage, DNase=dnase),
pamSiteOnly=TRUE,
margin=0.4)
```
## Comparing multiple GuideSets targeting the same region
The choice of the CRISPR nuclease can be influenced by the abundance of
PAM sequences recognized by a given nuclease in the target region.
For example, we would expect AT-rich regions to have fewer possible
targets for the SpCas9 nuclease, whose PAM is NGG. In these regions,
the CRISPR nuclease AsCas12a, whose PAM is TTTV, may prove more
appropriate. Given multiple `GuideSet`s targeting the same region,
we can compare the gRNAs of each in the same
plot using `plotMultipleGuideSets`.
Here, we pass our `GuideSet`s targeting an exon in the
human gene LTN1 in a named list. Note that there are no available
options for displaying guide labels or guide stacking, and only
the PAM sites are plotted. We will also add a track to monitor
the percent GC content (using a window roughly the
length of our protospacers). Not surprisingly, this AT-rich
region has fewer targets for SpCas9 compared
to AsCas12a. (Note: when plotting several GuideSets
you may need to increase the height of the plot space in
order for the track names to appear on the left side; see
"Setting plot size" below.)
We first load the precomputed `GuideSet` objects:
```{r}
data("cas9GuideSet", package="crisprViz")
data("cas12aGuideSet", package="crisprViz")
data("ltn1GeneModel", package="crisprViz")
```
```{r, fig.height=8}
plotMultipleGuideSets(list(SpCas9=cas9GuideSet, AsCas12a=cas12aGuideSet),
geneModel=ltn1GeneModel,
targetGene="LTN1",
bsgenome=BSgenome.Hsapiens.UCSC.hg38,
margin=0.2,
gcWindow=10)
```
# Setting plot size
Plots with many gene isoforms and/or gRNAs may require more space to render
than is allotted by your graphical device's default settings, resulting in an
error. One solution, depending on your graphical device, is offered by the
[**grDevices** package](https://www.rdocumentation.org/packages/grDevices/versions/3.6.2/topics/Devices).
Here is an example using macOS Quartz device:
```{r, eval=FALSE}
grDevices::quartz("Example plot", width=6, height=7)
# plot function
```
# Session Info
```{r}
sessionInfo()
```