Position-dependent sequence motif preferences of SpCas9 are largely determined by scaffold-complementary spacer motifs.

Streptococcus pyogenes Cas9 (SpCas9) nuclease exhibits considerable position-dependent sequence preferences. The reason behind these preferences is not well understood and is difficult to rationalise, since the protein establishes interactions with the target-spacer duplex in a sequence-independent manner. We revealed here that intramolecular interactions within the single guide RNA (sgRNA), between the spacer and the scaffold, cause most of these preferences. By using in cellulo and in vitro SpCas9 activity assays with systematically designed spacer and scaffold sequences and by analysing activity data from a large SpCas9 sequence library, we show that some long (>8 nucleotides) spacer motifs, that are complementary to the RAR unit of the scaffold, interfere with sgRNA loading, and that some motifs of more than 4 nucleotides, that are complementary to the SL1 unit, inhibit DNA binding and cleavage. Furthermore, we show that intramolecular interactions are present in the majority of the inactive sgRNA sequences of the library, suggesting that they are the most important intrinsic determinants of the activity of the SpCas9 ribonucleoprotein complex. We also found that in pegRNAs, sequences at the 3' extension of the sgRNA that are complementary to the SL2 unit are also inhibitory to prime editing, but not to the nuclease activity of SpCas9.

Primary pulmonary choriocarcinoma: a case report.

BACKGROUND: Primary pulmonary choriocarcinoma (PPC) is an extremely rare clinical entity. In contrast with gestational trophoblastic tumors that show an extreme sensitivity for chemotherapy, extragonadal choriocarcinomas are mostly unresponsive to surgical and chemotherapeutic treatment and are associated with poor prognosis. The reason non-gestational choriocarcinomas behave so differently from gestational tumors is unknown. CASE: In the present case we report a 30-year-old female patient with primary choriocarcinoma of lung localization who was successfully treated with surgical resection and multiple cycles of combination chemotherapy. During her recovery she was followed up by human chorionic gonadotropin (hCG) titer measurement, and after 1 year of close surveillance of beta-hCG levels her disease achieved complete remission. CONCLUSION: Because of the extreme rarity of this malignancy, there are no standardized therapeutic guidelines for treatment. The first choice in treatment of PPC is surgical resection. Postoperatively chemotherapy is indicated immediately, because definitive histologic diagnosis is not essential before chemotherapy since beta-hCG is a reliable tumor marker for choriocarcinoma.