Protocols: Optimized CRISPR system for genome editing in zebrafish

Publications

CRISPR-Cas9 protocol in Cold Spring Harbor Protocol

Optimized CRISPR-Cas9 System for Genome Editing in zebrafish

Charles E. Vejnar, Miguel A. Moreno-Mateos, Daniel Cifuentes, Ariel A. Bazzini, Antonio J. Giraldez

Cold Spring Harbor Protocols 2016

doi:10.1101/pdb.prot086850

PMID:27698232

The CRISPR-Cas9 protocol is introduced in:

Optimization Strategies for the CRISPR–Cas9 Genome-Editing System

Charles E. Vejnar, Miguel A. Moreno-Mateos, Daniel Cifuentes, Ariel A. Bazzini, Antonio J. Giraldez

Cold Spring Harbor Protocols 2016

doi:10.1101/pdb.top090894

PMID:27698246

CRISPR-Cas12a (Cpf1) protocol in Methods

Optimized CRISPR-Cpf1 system for genome editing in zebrafish

Juan P. Fernandez, Charles E. Vejnar, Antonio J. Giraldez, Romain Rouet, Miguel A. Moreno-Mateos

Methods 2018

doi:10.1016/j.ymeth.2018.06.014

PMID:29964176

Mut-Seq pipeline installation

1. Requirements

   • Python 3.2 (or more recent). A convenient Python installer like Anaconda can be used.
   • Download and install the GMAP read aligner.

2. Download Mut-Seq package. Do not unzip it; this Python ZIP application contains Mut-Seq script and dependencies and can be used with Python directly (see example below). If needed, source code is available under the MPLv2 licence.

Mut-Seq example

1. Download and unzip example source with:

   unzip -d mutseq_example example.zip
   

   This archive contains:

   1. loci.csv with the loci description
   2. seq_run1 folder with sequencing reads as FASTQ files
   3. output/report.txt is a precomputed report. A similar report should be obtained at the end of this tutorial.

2. Start Mut-Seq analysis with:

   python3 mutseq.pyz -l mutseq_example/loci.csv -d mutseq_example -o report.txt --processor 4 --min_allele_reads 10
   

   If you didn’t download mutseq.pyz (see previous section) to the current folder, change the path to mutseq.pyz in the command above.

3. While running, output should look like:

   14:10 - Parsing mutseq_example/loci.csv: 1 loci and 1 sample(s) found.
   14:10 - Creating index in .
   14:10 - Aligning seq_run1
   14:10 - Loading file(s): mutseq_example/seq_run1/AG00792_R1_001.fastq.gz
   14:10 - Starting GMAP (seq_run1,R1) with ['gmap', '--dir', '.', '--db', 'index', '--min-intronlength', '1600', '--allow-close-indels', '2', '--npaths', '1', '--ordered', '--input-buffer-size', '12000', '--batch', '4', '--format', 'samse', '--nthreads', '4']
   14:10 - Loading file(s): mutseq_example/seq_run1/AG00792_R2_001.fastq.gz
   14:10 - Starting GMAP (seq_run1,R2) with ['gmap', '--dir', '.', '--db', 'index', '--min-intronlength', '1600', '--allow-close-indels', '2', '--npaths', '1', '--ordered', '--input-buffer-size', '12000', '--batch', '4', '--format', 'samse', '--nthreads', '4']
   14:11 - Detecting allele in seq_run1
   14:11 - Writing report in report.txt
   

4. Results will be in report.txt. It should be identical to the report in output/ folder from the example archive.