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As an allotetraploid, most genes have multiple copies that belong to At and Dt subgenomes in cotton (Gossypium hirsutum). Allotetraploid cotton genome is very complex (AADD, 2n= 4x= 52) with a large genome size of 2.5 Gb. As a result, different types of genome editing tools are desirable to improve the functional genomic research in cotton. CRISPR/Cpf1 (Cas12a) is a novel member of the CRISPR/Cas system from Alicyclobacillus acidoterrestris, and currently, three kinds of Cpf1 are commonly used in genome editing, namely AsCpf1, LbCpf1 and FnCpf1. In 2015, type V CRISPR/Cpf1 (Cas12a) was applied in human cells to create gene knockout mutation for the first time (Zetsche et al., 2015). Several applications in rice showed that LbCpf1 exhibited higher genome editing efficiency than AsCpf1 and spCas9 (Tang et al., 2017, 2018). The LbCpf1 used in this study is from Lachnospiraceae bacterium ND2006 …