Background Cas12a (formerly known as Cpf1), the class II type V CRISPR nuclease, has been widely used for genome editing in mammalian cells and plants due to its distinct characteristics from Cas9. Despite being one of the most robust Cas12a nucleases, LbCas12a in general is less efficient than SpCas9 for genome editing in human cells, animals, and plants. Results To improve the editing efficiency of LbCas12a, we conduct saturation mutagenesis in E. coli and identify 1977 positive point mutations of LbCas12a. We selectively assess the editing efficiency of 56 LbCas12a variants in human cells, identifying an optimal LbCas12a variant (RVQ: G146R/R182V/E795Q) with the most robust editing activity. We further test LbCas12a-RV, LbCas12a-RRV, and LbCas12a-RVQ in plants and find LbCas12a-RV has robust editing activity in rice and tomato protoplasts. Interestingly, LbCas12a-RRV, resulting from the stacking of RV and D156R, displays improved editing efficiency in stably transformed rice and poplar plants, leading to up to 100% editing efficiency in T0 plants of both plant species. Moreover, this high-efficiency editing occurs even at the non-canonical TTV PAM sites. Conclusions Our results demonstrate that LbCas12a-RVQ is a powerful tool for genome editing in human cells while LbCas12a-RRV confers robust genome editing in plants. Our study reveals the tremendous potential of these LbCas12a variants for advancing precision genome editing applications across a wide range of organisms.

As synthetic biology becomes increasingly capable and accessible, it is likewise increasingly critical to be able to make accurate biosecurity determinations regarding the pathogenicity or toxicity of particular nucleic acid or amino acid sequences. At present, this is typically done using the BLAST algorithm to determine the best match with sequences in the NCBI nucleic acid and protein databases. Neither BLAST nor any of the NCBI databases, however, are actually designed for biosafety determination. Critically, taxonomic errors or ambiguities in the NCBI nucleic acid and protein databases can also cause errors in BLAST-based taxonomic categorization. With heavily studied taxa and frequently used biotechnology tools, even low frequency taxonomic categorization issues can lead to high rates of errors in biosecurity decision-making. Here we focus on the implications for false positives, finding that BLAST against NCBI’s protein database will now incorrectly categorize a number of commonly used biotechnology tool sequences as the pathogens or toxins with which they have been used. Paradoxically, this implies that problems are expected to be most acute for the pathogens and toxins of highest interest and for the most widely used biotechnology tools. We thus conclude that biosecurity tools should shift away from BLAST against general purpose databases and towards new methods that are specifically tailored for biosafety purposes.

As synthetic biology becomes increasingly capable and accessible, it is likewise increasingly critical to be able to make accurate biosecurity determinations regarding the pathogenicity or toxicity of particular nucleic acid or amino acid sequences. At present, this is typically done using the BLAST algorithm to determine the best match with sequences in the NCBI databases. Neither BLAST nor the NCBI databases, however, are actually designed for biosafety determination. Critically, taxonomic errors or ambiguities in the NCBI databases can also cause errors in BLAST-based taxonomic categorization. With heavily studied taxa and frequently used biotechnology tools, even low frequency taxonomic categorization issues can lead to high rates of errors in biosecurity decision-making. Here we focus on the implications for false positives, finding that NCBI BLAST will now incorrectly categorize a number of commonly used biotechnology tool sequences as the pathogens or toxins with which they have been used. Paradoxically, this implies that problems are expected to be most acute for the pathogens and toxins of highest interest and the most widely used biotechnology tools. We thus conclude that biosecurity tools should shift away from BLAST against NCBI and towards new methods that are specifically tailored for biosafety purposes.

1Using a method for pathogen screening in DNA synthesis orders, we have identified a 1 2 number of amino acid sequences that distinguish 2019-nCoV (Wuhan Coronavirus) from all 1 3 other known viruses in Coronaviridae. We find three main regions of unique sequence: two in 1 4 the 1ab polyprotein QHO60603.1, one in surface glycoprotein QHO60594.1.