CRISPR Designer

CRISPR Designer is a tool for the rapid design of guide sequences and repair templates to generate point mutations within 60 nucleotides of a PAM site in a target protein. The primers and sequences generated are designed for use with the protocol outlined by Ryan et al. 2016 titled, “Crispr–cas9 genome engineering in Saccharomyces cerevisiae cells”.

Dependencies for CRISPR designer can be found in requirements.txt, Python >=3.7 is also required.

Installation on MacOSX and Linux

At the command line, change directory to the directory where CRISPR-designer.py was downloaded, E.g , using the full path name.

cd <download-directory>

Now move the file to where you normally keep your binaries. This directory should be in your path. Note: you may require administrative privileges to do this (either switching user to root or by using sudo).

As root:

mv CRISPR-designer.py /usr/local/bin/

As regular user:

sudo mv CRISPR-designer.py /usr/local/bin/

After installation, CRISPR Designer can be run directly from the shell or Terminal using the following command:

CRISPR-designer.py

Alternatively, CRISPR Designer can be run from an IDE.

Using CRISPR Designer

Before using this tool please familiarise yourself with the protocol (see bibliography)

Inputs

CRISPR Designer requires the following inputs while running:

	CRISPR-designer.py --sequence sequence.fasta \
		--position 174 \
		--mutation A \
		--output output/directory

Arguments	Input
--sequence	Path to the FASTA file containing the Open Reading Frame of the target protein flanked by 300 nucleotides on each side. E.g 300bp-ORF-300bp
--position	Position of the amino acid to be mutated (e.g 174).
--mutation	Amino acid to subsititute at the target position as single letter code. (e.g A)
--output	(Optional) A directory to store the output file in. If no directory is provided the file will be saved to the current working directory

In addition, the following options are also available:

Option	Description
--help	Display help menu with usage information
--version	Display version information

Finding Correct Sequence Data

For CRISPR Designer to function and design all sequences ,correctly, the coding sequence of the protein and the surrounding 300 nucleotides eithe side of the open reading frame are required. CRISPR designer is not designed to implement mutations in genes containing introns.

The appropriate sequence for most genes can be accessed for a variety of yeasts from the Saccharomyces Genome Database using a custom retrieval.

Output

All data is written to a single output file. The file is named based on mutation generated by the user. E.g. If you mutated glutamine to alanine at position 140 of your protein, the output file would be E140A.txt .

Data is formatted similiar to a fasta file with each header displaying the sequence name and length in base pairs (n bp). Mutations to the provided sequence are highlighted by lower case lettering.

#########################
 Primers for Guide RNA 
#########################

> sgRNA forward primer 60 bp
sequence

> sgRNA reverse primer 60 bp
sequence

###############################
 Primers for Repair Template 
###############################

> repair template 60 bp
sequence

> forward repair template primer 70 bp
sequence

> reverse repair template primer 70 bp
sequence

> full repair template 160 bp
sequence

#########################
 Primers for Sequencing 
#########################

> amplified region (reference for sequencing) 350 bp
sequence 

> forward primer for amplified region 20 bp
sequence 

> reverse primer for amplified region 20 bp
sequence 

> primer for sequencing the amplified region 20 bp
sequence 

Notes on Function

CRISPR designer automatically designs sgRNA sequences, repair templates, and sequencing primers based on the inputs provided by the user. Codons are automatically mutated to implement the desired mutation and in the case of the 60mer protocol an additional synonymous mutation is also automatically introduced into the repair template.

If no appropriate PAM site can be found within 60 nucleotides region upstream of the target mutation, the programme will exit with no output. Although, It may still be possible to implement the mutation the on the reverse-strand or by using a larger search region upstream of nearby PAM sites. However, CRISPR-designer is currently unable to assist in these scenarios. (Please refer to the original protocol by Ryan et al., 2016)

Known Issues

1. Implementing a mutation more than 60 nucleotides away from a PAM site will result in CRISPR Designer stopping and designing no sequences.

Issues 1 is currently beyond the scope of CRISPR designer. Please refer to the Notes on Function section for the best solution to this issue.

Future Updates

Future updates may include:

• Support for Windows
• Reverse strand sequence designer to design for mutations more than 60 nucleotides away from a PAM site on the forward strand.
• Expansion of tool to include other methods outlined by Ryan et al. 2016 such as DNA bar coding (Please request if you’re interested!).

Feedback

Any and all feedback are welcome, just raise an issue, and I'll get back to you!

Bibliography

Ryan, O. W., Poddar, S., & Cate, J. H. D. (2016). Crispr–cas9 genome engineering in Saccharomyces cerevisiae cells. Cold Spring Harbor Protocols, 2016(6), 525–533. https://doi.org/10.1101/pdb.prot086827