Plant gene targeting (GT) can be utilized to precisely replace up to several kilobases of a plant genome. Recent studies using the powerful clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) nucleases significantly improved plant GT efficiency. However, GT for loci without associated selection markers is still inefficient. We previously utilized Lachnospiraceae bacterium Cas12a (LbCas12a) in combination with a replicon for tomato GT and obtained high GT efficiency with some selection markers. In this study, we customize and advance our GT system by using a temperature-tolerant LbCas12a (ttLbCas12a) in combination with various crRNA forms and chemical treatments to suppress the canonical non-homologous end-joining pathway in tomato. Our work demonstrates the significance of the selection of gene scissors, the appropriate design of LbCas12a gRNAs, the use of chemical treatments, and the establishment of favorable experimental conditions for further enhancement of plant HDR to enable efficient GT in tomato.