The CCTL (Cpf1-assisted Cutting and Taq DNA ligase-assisted Ligation) method for efficient editing of large DNA constructsin vitro

Abstract

As Cpf1 cleaves double-stranded DNA in a staggered way, it can be used in DNA assembly. However, the Cpf1 cleavage was found to be inaccurate, which may cause errors in DNA assembly. Here, the Cpf1 cleavage sites were precisely characterized, where the cleavage site on the target strand was around the 22nd base relative to the protospacer adjacent motif site, but the cleavage on the non-target strand was affected by the spacer length. When the spacer length was 20 nt or longer, Cpf1 mainly cleaved around the 14th and the 18th bases on the non-target strand; otherwise, with a shorter spacer (i.e. 17–19 nt), Cpf1 mainly cleaved after the 14th base, generating 8-nt sticky ends. With this finding, Cpf1 with a 17-nt spacer crRNA were employed for in vitro substitution of the actII-orf4 promoter in the actinorhodin biosynthetic cluster with a constitutively expressing promoter. The engineered cluster yielded more actinorhodin and produced actinorhodin from an earlier phase. Moreover, Taq DNA ligase was further employed to increase both the ligation efficiency and the ligation accuracy of the method. We expect this CCTL (Cpf1-assisted Cutting and Taq DNA ligase-mediated Ligation) method can be widely used in in vitro editing of large DNA constructs.

Transforming plant biology and breeding with CRISPR/Cas9, Cas12 and Cas13

“…1B) [64,65]. In contrast to Cas9, Cas12a solely requires a small crRNA to mediate its activity, target specificity is determined by a longer spacer, requiring at least 22 nt for maximum efficiency [64,66]. Instead of G-rich PAMs required by Cas9, Cas12a recognizes T-rich PAMs and thus further increases the number of potential target sites.…”

Improved CRISPR‐Cas12a‐assisted one‐pot DNA editing method enables seamless DNA editing

“…Previous work demonstrated that FnCas12a can perform DNA editing in vitro (Lei et al, ; S. Y. Li et al, ). However, limited PAM recognition restricts its application.…”

“…Previous work demonstrated that FnCas12a can perform DNA editing in vitro (Lei et al, 2017;S. Y. Li et al, 2016).…”

“…Among the characterized proteins in the Cas12a family, FnCas12a from Francisella novicida shows advantages in short PAM sequence recognition and preference to cleave the 18th to 23rd base distal from the PAM site, thus generating a 5 bp user‐defined sticky end (S. Y. Li, Zhao, & Wang, ). Therefore, the FnCas12a can sever as a Type II RE like digestion enzyme in molecular cloning, and it has already been demonstrated in the C‐brick standard and CCTL method (Lei et al, ; S. Y. Li et al, ). However, digestion and ligation are performed as separate time‐consuming steps in those methods, and fragment purification is required to eliminate repeated digestion and back ligation.…”

“…Li, Zhao, & Wang, 2016). Therefore, the FnCas12a can sever as a Type II RE like digestion enzyme in molecular cloning, and it has already been demonstrated in the C-brick standard and CCTL method (Lei et al, 2017;S. Y. Li et al, 2016).…”