title: ÐÐ·ÑÑÐµÐ½Ð¸Ðµ Ð°ÐºÑÐ¸Ð²Ð½Ð¾ÑÑÐ¸ Ð½ÑÐºÐ»ÐµÐ°Ð·Ñ CRISPR-Cas ÑÐ¸ÑÑÐµÐ¼Ñ V-A ÑÐ¸Ð¿Ð° Ð¸Ð· Ð±Ð°ÐºÑÐµÑÐ¸Ð¹ Sedimentisphacra cyanobacteriorum Ð² ÑÑÐºÐ°ÑÐ¸Ð¾ÑÐ¸ÑÐµÑÐºÐ¸Ñ ÐºÐ»ÐµÑÐºÐ°Ñ
title: Ð²ÑÐ¿ÑÑÐºÐ½Ð°Ñ ÐºÐ²Ð°Ð»Ð¸ÑÐ¸ÐºÐ°ÑÐ¸Ð¾Ð½Ð½Ð°Ñ ÑÐ°Ð±Ð¾ÑÐ° Ð±Ð°ÐºÐ°Ð»Ð°Ð²ÑÐ°
titleType: Subtitle
title: Study of the activity of the CRISPR-Cas nuclease of the V-A-type system from the Sedimentisphaera Cyanobacteriorum bacterium in eukaryotic cells
Creators: name: Ð§ÐµÑÐ½Ð¾Ð²Ð°, ÐÐµÑÐ¾Ð½Ð¸ÐºÐ°
Publisher: name: Ð¡Ð°Ð½ÐºÑ-ÐÐµÑÐµÑÐ±ÑÑÐ³ÑÐºÐ¸Ð¹ Ð¿Ð¾Ð»Ð¸ÑÐµÑÐ½Ð¸ÑÐµÑÐºÐ¸Ð¹ ÑÐ½Ð¸Ð²ÐµÑÑÐ¸ÑÐµÑ ÐÐµÑÑÐ° ÐÐµÐ»Ð¸ÐºÐ¾Ð³Ð¾
Year: 2023
Subjects: ÑÐµÐ´Ð°ÐºÑÐ¸ÑÐ¾Ð²Ð°Ð½Ð¸Ðµ Ð³ÐµÐ½Ð¾Ð¼Ð°
CRISPR-Cas
Cas12a
Sedimentisphaera cyanobacteriorum
ÐºÐ»ÐµÑÐºÐ¸ HEK293T
genome editing
HEK293T cells

description: ÐÐ°Ð½Ð½Ð°Ñ ÑÐ°Ð±Ð¾ÑÐ° Ð¿Ð¾ÑÐ²ÑÑÐµÐ½Ð° Ð¸ÑÑÐ»ÐµÐ´Ð¾Ð²Ð°Ð½Ð¸Ñ Ð°ÐºÑÐ¸Ð²Ð½Ð¾ÑÑÐ¸ Ð½ÑÐºÐ»ÐµÐ°Ð·Ñ ScCas12a Ð² ÐºÐ»ÐµÑÐºÐ°Ñ HEK293T. ÐÐ°Ð´Ð°ÑÐ¸, ÐºÐ¾ÑÐ¾ÑÑÐµ ÑÐµÑÐ°Ð»Ð¸ÑÑ Ð² ÑÐ¾Ð´Ðµ Ð¸ÑÑÐ»ÐµÐ´Ð¾Ð²Ð°Ð½Ð¸Ñ: - ÐÐ·ÑÑÐ¸ÑÑ Ð»Ð¸ÑÐµÑÐ°ÑÑÑÐ½ÑÐµ Ð¸ÑÑÐ¾ÑÐ½Ð¸ÐºÐ¸ Ð¿Ð¾ Ð´Ð°Ð½Ð½Ð¾Ð¹ ÑÐµÐ¼Ðµ; - Ð¡Ð¾Ð·Ð´Ð°ÑÑ Ð³ÐµÐ½ÐµÑÐ¸ÑÐµÑÐºÑÑ ÐºÐ¾Ð½ÑÑÑÑÐºÑÐ¸Ñ, Ð½ÐµÑÑÑÑÑ Ð½Ð°ÑÐ¸Ð²Ð½ÑÑ Ð¿Ð¾ÑÐ»ÐµÐ´Ð¾Ð²Ð°ÑÐµÐ»ÑÐ½Ð¾ÑÑÑ Ð½ÑÐºÐ»ÐµÐ°Ð·Ñ ScCas12a Ð¸ ÐºÑÐ ÐÐ; - Ð¡Ð¾Ð·Ð´Ð°ÑÑ RNP-ÐºÐ¾Ð¼Ð¿Ð»ÐµÐºÑ CRISPR-ScCas12a; - ÐÑÐ¾Ð²ÐµÑÐ¸ÑÑ Ð°ÐºÑÐ¸Ð²Ð½Ð¾ÑÑÑ Ð½ÑÐºÐ»ÐµÐ°Ð·Ñ ScCas12a Ð² ÐºÐ»ÐµÑÐºÐ°Ñ ÑÐµÐ»Ð¾Ð²ÐµÐºÐ°. ÐÐ±ÑÐµÐºÑ Ð¸ÑÑÐ»ÐµÐ´Ð¾Ð²Ð°Ð½Ð¸Ñ â ÐºÐ»ÐµÑÐºÐ¸ HEK293T, ÑÑÐ°Ð½ÑÑÐµÑÐ¸ÑÐ¾Ð²Ð°Ð½Ð½ÑÐµ Ð´Ð²ÑÐ¼Ñ ÑÐ¿Ð¾ÑÐ¾Ð±Ð°Ð¼Ð¸: - ÐÑÐ¿Ð¾Ð»ÑÐ·Ð¾Ð²Ð°Ð½Ð¸Ðµ Ð¿Ð»Ð°Ð·Ð¼Ð¸Ð´Ñ, ÐºÐ¾ÑÐ¾ÑÐ°Ñ Ð½ÐµÑÑÑ: Ð³ÐµÐ½ Ð½Ð°ÑÐ¸Ð²Ð½Ð¾Ð¹ Ð¿Ð¾ÑÐ»ÐµÐ´Ð¾Ð²Ð°ÑÐµÐ»ÑÐ½Ð¾ÑÑÐ¸ Cas12 Ð±ÐµÐ»ÐºÐ° V-A ÑÐ¸Ð¿Ð°, Ð¾Ð±Ð½Ð°ÑÑÐ¶ÐµÐ½Ð½Ð¾Ð³Ð¾ Ð² Ð±Ð°ÐºÑÐµÑÐ¸Ð¸ Sedimentisphaera cyanobacteriorum, Ð¿Ð¾Ð´ ÑÐµÐ³ÑÐ»ÑÑÐ¸ÐµÐ¹ CMV Ð¿ÑÐ¾Ð¼Ð¾ÑÐ¾ÑÐ° Ñ NLS Ð½Ð° ÐºÐ¾Ð½ÑÐ°Ñ; Ð¿Ð¾ÑÐ»ÐµÐ´Ð¾Ð²Ð°ÑÐµÐ»ÑÐ½Ð¾ÑÑÑ Ð³ÐµÐ½Ð° EGFP, Ð¿Ð¾Ð·Ð²Ð¾Ð»ÑÑÑÐµÐ³Ð¾ Ð´ÐµÑÐµÐºÑÐ¸ÑÐ¾Ð²Ð°ÑÑ ÑÐºÑÐ¿ÑÐµÑÑÐ¸Ñ Ð±ÐµÐ»ÐºÐ° in vivo; - Ð¡Ð¾Ð·Ð´Ð°Ð½Ð¸Ðµ RNP-ÐºÐ¾Ð¼Ð¿Ð»ÐµÐºÑÐ°, ÑÐ¾ÑÑÐ¾ÑÑÐµÐ³Ð¾ Ð¸Ð· ScCas12a Ð½Ð°ÑÐ¸Ð²Ð½Ð¾Ð¹ Ð¿Ð¾ÑÐ»ÐµÐ´Ð¾Ð²Ð°ÑÐµÐ»ÑÐ½Ð¾ÑÑÐ¸ Ð½ÑÐºÐ»ÐµÐ°Ð·Ñ Ð¸ Ð³Ð¸Ð´Ð¾Ð²Ð¾Ð¹ Ð ÐÐ. Ð Ð°Ð±Ð¾ÑÐ° Ð²ÑÐ¿Ð¾Ð»Ð½ÐµÐ½Ð° Ð½Ð° Ð±Ð°Ð·Ðµ ÐÐ°ÑÑÐ½Ð¾-Ð¸ÑÑÐ»ÐµÐ´Ð¾Ð²Ð°ÑÐµÐ»ÑÑÐºÐ¾Ð³Ð¾ ÐºÐ¾Ð¼Ð¿Ð»ÐµÐºÑÐ° Â«ÐÐ°Ð½Ð¾Ð±Ð¸Ð¾ÑÐµÑÐ½Ð¾Ð»Ð¾Ð³Ð¸Ð¹Â» Ð¡Ð°Ð½ÐºÑ-ÐÐµÑÐµÑÐ±ÑÑÐ³ÑÐºÐ¾Ð³Ð¾ Ð¿Ð¾Ð»Ð¸ÑÐµÑÐ½Ð¸ÑÐµÑÐºÐ¾Ð³Ð¾ ÑÐ½Ð¸Ð²ÐµÑÑÐ¸ÑÐµÑÐ° ÐÐµÑÑÐ° ÐÐµÐ»Ð¸ÐºÐ¾Ð³Ð¾. ÐÑÐ¸ Ð½Ð°Ð¿Ð¸ÑÐ°Ð½Ð¸Ð¸ Ð»Ð¸ÑÐµÑÐ°ÑÑÑÐ½Ð¾Ð³Ð¾ Ð¾Ð±Ð·Ð¾ÑÐ° Ð¸ÑÐ¿Ð¾Ð»ÑÐ·Ð¾Ð²Ð°Ð»Ð¸ÑÑ ÑÐ»ÐµÐ´ÑÑÑÐ¸Ðµ Ð±Ð°Ð·Ñ Ð´Ð°Ð½Ð½ÑÑ: ScienceDirect, National Center of Biotechnology Information, PubMed Central, FEBS Press, Cyberleninka, Ð­Ð»ÐµÐºÑÑÐ¾Ð½Ð½Ð°Ñ Ð±Ð¸Ð±Ð»Ð¸Ð¾ÑÐµÐºÐ° Ð¡ÐÐ±ÐÐ£. ÐÐ¸Ð·Ð°Ð¹Ð½ Ð¿Ð»Ð°Ð·Ð¼Ð¸Ð´ Ð¸ Ð¿Ð¾Ð´Ð±Ð¾Ñ Ð¿ÑÐ°Ð¹Ð¼ÐµÑÐ¾Ð² Ð¾ÑÑÑÐµÑÑÐ²Ð»ÑÐ»ÑÑ Ñ Ð¸ÑÐ¿Ð¾Ð»ÑÐ·Ð¾Ð²Ð°Ð½Ð¸ÐµÐ¼ Ð¾Ð½Ð»Ð°Ð¹Ð½-Ð¿Ð»Ð°ÑÑÐ¾ÑÐ¼Ñ Â«Benchling (Benchling, Ð¡Ð¨Ð)Â». ÐÐ½Ð°Ð»Ð¸Ð· ÑÐµÐ·ÑÐ»ÑÑÐ°ÑÐ¾Ð² ÑÐ»ÐµÐºÑÑÐ¾ÑÐ¾ÑÐµÑÐ¸ÑÐµÑÐºÐ¾Ð³Ð¾ ÑÐ°Ð·Ð´ÐµÐ»ÐµÐ½Ð¸Ñ Ð½ÑÐºÐ»ÐµÐ¸Ð½Ð¾Ð²ÑÑ ÐºÐ¸ÑÐ»Ð¾Ñ Ð¿ÑÐ¾Ð¸Ð·Ð²Ð¾Ð´Ð¸Ð»Ð¸ Ð¿ÑÐ¸ Ð¿Ð¾Ð¼Ð¾ÑÐ¸ Ð¿ÑÐ¾Ð³ÑÐ°Ð¼Ð¼Ð½Ð¾Ð³Ð¾ Ð¾Ð±ÐµÑÐ¿ÐµÑÐµÐ½Ð¸Ñ Â«Imagelab (Bio Rad, Ð¡Ð¨Ð)Â». Ð ÑÐ¾Ð´Ðµ Ð¸ÑÑÐ»ÐµÐ´Ð¾Ð²Ð°Ð½Ð¸Ñ Ð±ÑÐ»Ð° Ð¿ÑÐ¾Ð²ÐµÑÐµÐ½Ð° Ð°ÐºÑÐ¸Ð²Ð½Ð¾ÑÑÑ Ð½ÑÐºÐ»ÐµÐ°Ð·Ñ ScCas12a Ð² ÐºÐ»ÐµÑÐºÐ°Ñ ÑÐµÐ»Ð¾Ð²ÐµÐºÐ°.
description: The work is dedicated to investigating the activity of ScCas12a nuclease in HEK293T cells. The tasks of the research: - Study the literary sources of the topic; - Create a genetic construct containing the native sequence of ScCas12a nuclease and crRNA; - Create the RNP-complex CRISPR-ScCas12a; - Research of ScCas12a nuclease activity in human cells. - The object of the research is HEK293T cells transfected in two ways: The use of a plasmid that contains: the gene of the native sequence Cas12 V-A type protein found in the bacterium Sedimentisphaera cyanobacterio-rum, under the regulation of a CMV promoter with NLS at the ends; the sequence of the EGFP gene, which allows detecting protein expression in vivo; - Creation of the RNP-complex consisting of ScCas12a native sequence of nuclease and guide RNA. The work was carried out in the analytical center of nano- and biotechnologies of Peter the Great St. Petersburg Polytechnic University. When writing the literature review, the following databases were used: Sci-enceDirect, National Center of Biotechnology Information, PubMed Central, FEBS Press, Cyberleninka, Polytech Electronic library. Plasmid design and selection of primers was carried out using the online platform Benchling (Benchling, USA). The analysis of the results of electrophoretic separation of nucleic acids was performed using the Imagelab (Bio Rad, USA) software. In the research, ScCas12a nuclease activity was studied in human cells.