The RNA-guided Cas12a nuclease forms a complex with a CRISPR RNA (crRNA) to cleave the double-stranded DNA target. Among others, Cas12a protein from Lachnospiraceae bacterium (LbCas12a) is widely used for biomedical research. For target recognition, LbCas12a requires a specific nucleotide sequence, named a protospacer adjacent motif (PAM). Besides the canonical TTTV PAM, LbCas12a can recognize other suboptimal PAMs. We examined a novel TTAA PAM for the LbCas12a nuclease and found that the specificity of cleavage was increased. We found that single nucleotide substitutions at all positions of the guide RNA except the 20th position blocked the cleavage of the target DNA. The type of nucleotide substitutions (U-A, U-C or U-G) did not affect the efficiency of cleavage in the 20th position. When we used the canonical PAM under the same conditions, we observed the cleavage of target DNA by LbCas12a in many positions, showing less specificity in given conditions. The efficiency and specificity of the LbCas12a nuclease were evaluated both by gel-electrophoresis and using FAM-labeled single-stranded probes. We were able to assess the change in fluorescence intensity only for several variants of guide RNAs. High specificity allows us to type single nucleotide substitutions and small deletions/insertions (1–2 nucleotides) and look for target mutations when knocking out.

Preeclampsia is a multisystem pregnancy disorder that occurs after 20 weeks of gestation, leading to e.g. preterm labor. It is characterized by hypertension, proteinuria, edema, and multiple organ dysfunction. Up to 8% of pregnancies are complicated by preeclampsia, which is one of the most serious causes of maternal and perinatal mortality [1]. For research of pregnancy disorders and development of therapy for it, a mouse model can be used due of the fact that pregnancy development in mice, especially at early stages, is somewhat similar to human and is well-studied, in particular, in terms of molecular biology [2]. One of the possible options for creating mouse models of preeclampsia is considered to be a mutation in the COMT gene encoding сatechol-O-methyltransferase [3]. This enzyme plays an important role in the catecholamines conversion and it also catalyzes the O-methylation of hydroxyestradiol producing methoxyestradiol. COMT gene knockout results in a phenotype similar to preeclampsia with elevated blood pressure and proteinuria [3]. The previous model was obtained through classic transgenesis methods with Neomycin cassette insertion in the COMT locus potentially influencing the results of the experiments. The development of the genome editing systems and its active utilization at Saint Petersburg State University made it possible to obtain a COMT-KO mouse line using CRISPR/Cas9 technology which had not been done in Russia before. This model will allow to effectively study the development of preeclampsia and ways to prevent and treat it. This work was supported by a Saint Petersburg State University grant for the development of scientific research (ID 92561695).