In silico analysis of guide designs for efficient CRISPR-Cpf1 gene editing in cyanobacteria

Abstract

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology has been extensively used as a promising approach for genome editing in a variety of organisms. Cas9 and Cpf1 are the two main CRISPR-associated endonucleases currently used in genome engineering. However, even though Cpf1 can be more efficient and reliable than Cas9 in some organisms, it has not been as extensively developed. Among the many microbial biotechnology platforms being studied today cyanobacteria are one of the most interesting, given their ability to convert CO2 to multiple desired products using only solar energy. Even though they have been employed in several genetic engineering studies, their relatively slow growth rate and the current low efficiency of CRISPR-based protocols present severe obstacles impairing their efficient development as biotechnological chassis. Recently, the CRISPR-Cpf1 system was shown to overcome several of the caveats encountered by Cas9-based techniques in cyanobacteria. In this work, an in silico analysis of constructs for efficient Cpf1-based genome editing in the marine cyanobacterium Synechococcus sp. PCC 7002 has been performed, to design efficient PAM sites and to validate the most efficient guide design. As a result of this study, we have designed several different constructs for genome engineering in this chassis strain, forming the basis of future practical studies on the subject.