DESCRIPTION

The CRISPR Tools pipeline assists in the prediction and prioritization of guides to be used in either a gene knock-out (KO) or knock-in (KI) project. Guide sequences are identified based upon the generalized form of N20NGG for use with S. pyogenes Cas9. Efficiency scores are assigned based upon the scoring algorithms described in Doench et al. 2014 and Moreno-Mateos et al. 2015 and off-target analysis is obtained through the use of Sanger CRISPR-Analyser (Hodgkins et al. 2015). The gene KO and KI pipelines provide all of the essential information necessary to perform a CRISPR/Cas9 genome editing experiment.

Gene Knock-out

The use of CRISPR/Cas9 genome editing has quickly become the favored approach for generating gene knock-outs. Here, we have implemented an approach to generate either whole exon deletions or internal exon deletions in order to facilitate detection of the genetic lesion by standard PCR. The current version of the program supports Ensembl annotations of the mouse genome and takes as input Ensembl exon IDs (e.g. ENSMUSE00000292922). The tool supports batch input and paired exon deletions where two exon IDs are comma separated (e.g. ENSMUSE00001268302,ENSMUSE00001259979). The user has the option to run in two modes: whole exon deletion (default) and internal deletion. Additional options include searching the masked or non-masked genome and having oligo sequences returned with the optimized stem loop adaptor described in Chen et al. 2013.

Exon deletions are designed by searching the flanking sequence (50-300 bp) of the critical exon to identify guides. The identified guides are subsequently filtered to select guides that map uniquely to the mouse genome but allow a mismatch tolerance of 3 or more {0:1; 1:0; 2:0}. Guides that map with one or two mismatches are removed. Additionally, guides that collide with a neighbor exon are also eliminated. The remaining guides are subsequently rank ordered based upon their efficiency scores and the top two non-overlapping guides upstream and downstream of the critical exon are returned. Paired exon deletions are achieved using the same approach whereby the top two upstream guides of the first exon and the top two downstream guides of the second exon are selected.

Internal exon deletions are appropriate for gene models where all of the exons are in the same phase or the gene has one or two large coding exons. Guides within the exon are prioritized as above and the top two upstream and downstream guides that meet a minimum 50 bp separation distance are returned.

Suggestions for failed designs include: (1) increase flanking distance; (2) select alternate exon; (3) search non-masked genome; (4) reduce mismatch tolerance; (5) try internal exon deletion

Each run returns a zipped results file containing the following outputs:

Gene Knock-in

We have developed a CRISPR Gene KI tool to assist in the identification of guides centered on a specific genomic coordinate. Guides within +/- 50 bp of the point of interest are ordered according to their position relative to the desired insertion site and predicted efficiency scores are provided. In addition, the off-target analysis is returned for each guide. This program is useful for designing nucleotide substitution experiments or gene insertion experiments.

Each run returns a zipped results file containing the following outputs:

Sequence_csv = A list of gene target names with the point mutation position (e.g. chr7:28776872) listed, the point mutation change (i.e. A>G), sequence region of 50 bases on either side of the point mutation, the nucleotide sequence of the 100 bases and number of guides the tool identified for each gene target.

KI_guides.csv = summary of gene target with the point mutation position followed by the spacer sequence and filtered due to mismatch with both BROAD and CRISPRscan scores followed by a category 1-4 which indicates our prioritization of the guide based on proximity to the mutation site. Guides that fall into category 1 are highest priority.

Guide Prioritization Categories:

  1. The protospacer adjacent motif -PAM (NGG) overlaps the mutation site, so that by mutating one of the G’s the PAM sequence will be unable to recruit the guide to its specific target, effectively destroying the site. The second G is the most critical to the PAM sequence as it is somewhat possible, but not probable for a NaG to still allow the guide to bind.
  2. The point mutation is within the first 1-3 bases of the guide, either at the cut site or right next to it. With point mutation replacement the closer the cut site is to the base to be altered the better.
  3. Within the first 10 bases of the guide to the cut site.
  4. ll others, including mutation site on the wrong side of the PAM and cut site. This is not very useful.

REFERENCES

Chen, B., Gilbert, L., Cimini, B., Schnitzbauer, J., Zhang, W., Li, G.-W., Park, J., Blackburn, E., Weissman, J., Qi, L., et al. (2013). Dynamic Imaging of Genomic Loci in Living Human Cells by an Optimized CRISPR/Cas System. Cell 155.

Doench, J., Hartenian, E., Graham, D., Tothova, Z., Hegde, M., Smith, I., Sullender, M., Ebert, B., Xavier, R., and Root, D. (2014). Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation. Nat. Biotechnol. 32, 1262–1267.

Hodgkins, A., Farne, A., Perera, S., Grego, T., Parry-Smith, D.J., Skarnes, W.C., and Iyer, V. (2015). WGE: a CRISPR database for genome engineering. Bioinformatics 31, 3078–3080.

Moreno-Mateos, M.A., Vejnar, C.E., Beaudoin, J.-D.D., Fernandez, J.P., Mis, E.K., Khokha, M.K., and Giraldez, A.J. (2015). CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo. Nat. Methods 12, 982–988.

CITATION

Please cite this tool as:

Peterson KA, Beane GL, Goodwin LO, Kutny PM, Reinholdt LG, Murray SA. CRISPRtools: a flexible computational platform for performing CRISPR/Cas9 experiments in the mouse. Mamm Genome. 2017 Mar 9 doi:10.1007/s00335-017-9681-z


CONTRIBUTORS

Kevin A. Peterson, Ph.D.
Glen Beane, M.S.
Leslie Goodwin, Ph.D.
Laura Reinholdt, Ph.D.
Stephen A. Murray, Ph.D.

AKNOWLEDGEMENTS

This work was funded by support from High Throughput Production and Cryopreservation of Knockout Mice (OD011185) to S.A.M., and the Mouse Mutant Resource and Research Center grant (OD010972) to L.G.R.

REFERENCE DATA

CRISPR-Tools obtains flanking sequence from mouse reference genome GRCm38

Exon data is obtained monthly from the Ensembl Biomart.

Andrew Yates, Wasiu Akanni, M. Ridwan Amode, Daniel Barrell, Konstantinos Billis, Denise Carvalho-Silva, Carla Cummins, Peter Clapham, Stephen Fitzgerald, Laurent Gil1 Carlos Garcín Girón, Leo Gordon, Thibaut Hourlier, Sarah E. Hunt, Sophie H. Janacek, Nathan Johnson, Thomas Juettemann, Stephen Keenan, Ilias Lavidas, Fergal J. Martin, Thomas Maurel, William McLaren, Daniel N. Murphy, Rishi Nag, Michael Nuhn, Anne Parker, Mateus Patricio, Miguel Pignatelli, Matthew Rahtz, Harpreet Singh Riat, Daniel Sheppard, Kieron Taylor, Anja Thormann, Alessandro Vullo, Steven P. Wilder, Amonida Zadissa, Ewan Birney, Jennifer Harrow, Matthieu Muffato, Emily Perry, Magali Ruffier, Giulietta Spudich, Stephen J. Trevanion, Fiona Cunningham, Bronwen L. Aken, Daniel R. Zerbino, Paul Flicek; Ensembl 2016. Nucleic Acids Res. 2016 44 Database issue:D710-6. PubMed PMID: 26687719; PubMed CentralPMCID: PMC4702834. doi: 10.1093/nar/gkv1157

System Update Information

CRISPR tools is unavailable during scheduled maintenance at 3:00AM-4:00AM on the first day of every month. It is reccomended that you do not schedule a task until after the update is complete, or too close to when it begins.

Last update was completed on Dec 01 2017 08:00 AM UTC