Abstract
The CRISPR/Cas9 system is a powerful, revolutionary tool for genome editing. However, it is not without limitations. There are PAM-free and CRISPR-tolerant regions that cannot be modified by the standard CRISPR/Cas9 system, and off-target activity impedes its broader applications. To avoid these drawbacks, we developed a very simple CRISPR/Cas9-assisted gRNA-free one-step (CAGO) genome editing technique which does not require the construction of a plasmid to express a specific gRNA. Instead, a universal N20 sequence with a very high targeting efficiency is inserted into the E. coli chromosome by homologous recombination, which in turn undergoes a double-stranded break by CRISPR/Cas9 and induces an intra-chromosomal recombination event to accomplish the editing process. This technique was shown to be able to edit PAM-free and CRISPR-tolerant regions with no off-target effects in Escherichia coli. When applied to multi-locus editing, CAGO was able to modify one locus in two days with a near 100% editing efficiency. Furthermore, modified CAGO was used to edit large regions of up to 100 kbp with at least 75% efficiency. Finally, genome editing by CAGO only requires a transformation procedure and the construction of a linear donor DNA cassette, which was further simplified by applying a modular design strategy. Although the technique was established in E. coli, it should be applicable to other organisms with only minor modifications.
Highlights
Genome editing technology is used for rationally designed chromosome modification, which is one of the most significant techniques in molecular biology and plays an important role in many biotechnological applications
A universal N20 sequence with optimal targeting efficiency is inserted into the chromosome by homologous recombination, which in turn causes a double-strand break via CRISPR/Cas[9], and induces an intra-chromosomal recombination event to accomplish the editing process
The genome-editing procedure was implemented via a designed DNA-editing cassette, which contained three homology arms of the target locus (L, L_short and R), an insertion fragment if necessary, a selection marker, and a CRISPR/Cas[9] recognition region (N20PAM)
Summary
Design and development of the CRISPR/Cas9-assisted gRNA-free one-step (CAGO) genome editing technique. The genome-editing procedure was implemented via a designed DNA-editing cassette, which contained three homology arms of the target locus (L, L. short and R), an insertion fragment if necessary, a selection marker (such as chloramphenicol resistance), and a CRISPR/Cas[9] recognition region (N20PAM). This universal N20PAM was designed and selected to have minimal sequence similarity with all N20PAM sequences in the target genome, in order to guarantee the lowest possible off-target frequency. The First-round recombination efficiency of this DNA cassette was determined to be 298.3 ± 27.2
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