SMAP design: a multiplex PCR amplicon and gRNA design tool to screen for natural and CRISPR-induced genetic variation.
Journal
Nucleic acids research
ISSN: 1362-4962
Titre abrégé: Nucleic Acids Res
Pays: England
ID NLM: 0411011
Informations de publication
Date de publication:
24 04 2023
24 04 2023
Historique:
accepted:
12
01
2023
revised:
14
12
2022
received:
16
07
2022
medline:
25
4
2023
pubmed:
1
2
2023
entrez:
31
1
2023
Statut:
ppublish
Résumé
Multiplex amplicon sequencing is a versatile method to identify genetic variation in natural or mutagenized populations through eco-tilling or multiplex CRISPR screens. Such genotyping screens require reliable and specific primer designs, combined with simultaneous gRNA design for CRISPR screens. Unfortunately, current tools are unable to combine multiplex gRNA and primer design in a high-throughput and easy-to-use manner with high design flexibility. Here, we report the development of a bioinformatics tool called SMAP design to overcome these limitations. We tested SMAP design on several plant and non-plant genomes and obtained designs for more than 80-90% of the target genes, depending on the genome and gene family. We validated the designs with Illumina multiplex amplicon sequencing and Sanger sequencing in Arabidopsis, soybean, and maize. We also used SMAP design to perform eco-tilling by tilling PCR amplicons across nine candidate genes putatively associated with haploid induction in Cichorium intybus. We screened 60 accessions of chicory and witloof and identified thirteen knockout haplotypes and their carriers. SMAP design is an easy-to-use command-line tool that generates highly specific gRNA and/or primer designs for any number of loci for CRISPR or natural variation screens and is compatible with other SMAP modules for seamless downstream analysis.
Identifiants
pubmed: 36718951
pii: 7016455
doi: 10.1093/nar/gkad036
pmc: PMC10123101
doi:
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
e37Informations de copyright
© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.
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