Abstract Summary CRISPR-Local is a high-throughput local tool for designing single-guide RNAs (sgRNAs) in plants and other organisms that factors in genetic variation and is optimized to generate genome-wide sgRNAs. CRISPR-Local outperforms other sgRNA design tools in the following respects: (i) designing sgRNAs suitable for non-reference varieties; (ii) screening for sgRNAs that are capable of simultaneously targeting multiple genes; (iii) saving computational resources by avoiding repeated calculations from multiple submissions and (iv) running offline, with both command-line and graphical user interface modes and the ability to export multiple formats for further batch analysis or visualization. We have applied CRISPR-Local to 71 public plant genomes, using both CRISPR/Cas9 and CRISPR/cpf1 systems. Availability and implementation CRISPR-Local can be freely downloaded from http://crispr.hzau.edu.cn/CRISPR-Local/. Supplementary information Supplementary data are available at Bioinformatics online.