title: ÐÐ¾Ð»ÑÑÐµÐ½Ð¸Ðµ Ð¸ Ñ Ð°ÑÐ°ÐºÑÐµÑÐ¸Ð·Ð°ÑÐ¸Ñ ÐºÐ°ÑÐ°Ð»Ð¸ÑÐ¸ÑÐµÑÐºÐ¸ Ð½ÐµÐ°ÐºÑÐ¸Ð²Ð½Ð¾Ð³Ð¾ ÑÑÑÐµÐºÑÐ¾ÑÐ¾Ð² ÑÐ¸ÑÑÐµÐ¼Ñ CRISPR/cas12d1
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: Obtaining and characterization of catalytically inactive effectors of the CRISPR/Cas12d1 system
Creators: name: ÐÐ»Ð¸Ð¼ÐºÐ¾, ÐÐ°Ð»ÐµÑÐ¸Ð¹
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: CRISPR
CRISPRi
Cas12d
Ð¸Ð½Ð°ÐºÑÐ¸Ð²Ð¸ÑÐ¾Ð²Ð°Ð½Ð½ÑÐ¹ ÐºÐ°ÑÐ°Ð»Ð¸ÑÐ¸ÑÐµÑÐºÐ¸Ð¹ Ð´Ð¾Ð¼ÐµÐ½
RuvC
Ñ Ð°ÑÐ°ÐºÑÐµÑÐ¸Ð·Ð°ÑÐ¸Ñ in vitro
inactivated catalytic domain
in vitro characterization

description: ÐÐ°Ð½Ð½Ð°Ñ ÑÐ°Ð±Ð¾ÑÐ° Ð¿Ð¾ÑÐ²ÑÑÐµÐ½Ð° Ð¿Ð¾Ð»ÑÑÐµÐ½Ð¸Ñ Ð¸ ÑÐ°ÑÐ°ÐºÑÐµÑÐ¸Ð·Ð°ÑÐ¸Ð¸ ÐºÐ°ÑÐ°Ð»Ð¸ÑÐ¸ÑÐµÑÐºÐ¸ Ð½ÐµÐ°ÐºÑÐ¸Ð²Ð½ÑÑ ÑÑÑÐµÐºÑÐ¾ÑÐ¾Ð² ÑÐ¸ÑÑÐµÐ¼Ñ CRISPR/Cas12d1 Ñ Ð¿Ð¾Ð¼Ð¾ÑÑÑ Ð²Ð½ÐµÑÐµÐ½Ð¸Ñ Ð¼Ð¸ÑÑÐµÐ½Ñ-Ð¼ÑÑÐ°ÑÐ¸Ð¹ Ð² ÑÑÐ½ÐºÑÐ¸Ð¾Ð½Ð°Ð»ÑÐ½ÑÐ¹ Ð½ÑÐºÐ»ÐµÐ°Ð·Ð½ÑÐ¹ Ð´Ð¾Ð¼ÐµÐ½ RuvC. Ð¦ÐµÐ»Ñ ÑÐ°Ð±Ð¾ÑÑ: Ð¿Ð¾Ð»ÑÑÐµÐ½Ð¸Ðµ Ð¸ ÑÐ°ÑÐ°ÐºÑÐµÑÐ¸Ð·Ð°ÑÐ¸Ñ ÐºÐ°ÑÐ°Ð»Ð¸ÑÐ¸ÑÐµÑÐºÐ¸ Ð½ÐµÐ°ÐºÑÐ¸Ð²Ð½ÑÑ ÑÑÑÐµÐºÑÐ¾ÑÐ¾Ð² ÑÐ¸ÑÑÐµÐ¼Ñ CRISPR/Cas12d1 (CasY1). 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: 1. ÐÑÐ¾Ð¸Ð·Ð²ÐµÐ´ÐµÐ½Ð¸Ðµ Ð²ÑÑÐ°Ð²Ð½Ð¸Ð²Ð°Ð½Ð¸Ñ Ð³ÐµÐ½ÐµÑÐ¸ÑÐµÑÐºÐ¸Ñ Ð¿Ð¾ÑÐ»ÐµÐ´Ð¾Ð²Ð°ÑÐµÐ»ÑÐ½Ð¾ÑÑÐ¸ ÑÐ¸ÑÑÐµÐ¼Ñ Cas12d1 Ð½Ð° Ð¸Ð·Ð²ÐµÑÑÐ½ÑÐµ Ð¿Ð¾ÑÐ»ÐµÐ´Ð¾Ð²Ð°ÑÐµÐ»ÑÐ½Ð¾ÑÑÐ¸ Cas12a Ð¸ Cas12e. ÐÐ¿ÑÐµÐ´ÐµÐ»ÐµÐ½Ð¸Ðµ ÑÑÐ½ÐºÑÐ¸Ð¾Ð½Ð°Ð»ÑÐ½Ð¾Ð³Ð¾ Â  Â  Ð´Ð¾Ð¼ÐµÐ½Ð° ÑÐ¸ÑÑÐµÐ¼Ñ Cas12d1 Ð¸ Ð¿Ð¾ÑÐµÐ½ÑÐ¸Ð°Ð»ÑÐ½ÑÐµ Ð¼ÑÑÐ°ÑÐ¸Ð¸, ÑÐ¿Ð¾ÑÐ¾Ð±Ð½ÑÐµ Ð¿ÑÐ¸Ð²ÐµÑÑÐ¸ Ðº ÑÑÑÐ°ÑÐµ Ð¸Ð¼ ÐºÐ°ÑÐ°Ð»Ð¸ÑÐ¸ÑÐµÑÐºÐ¾Ð¹ Ð°ÐºÑÐ¸Ð²Ð½Ð¾ÑÑÐ¸; 2.ÐÐ¾Ð´ÐµÐ»Ð¸ÑÐ¾Ð²Ð°Ð½Ð¸Ðµ Ð¸ ÑÐ±Ð¾ÑÐºÐ° Ð³ÐµÐ½ÐµÑÐ¸ÑÐµÑÐºÐ¸Ñ ÐºÐ¾Ð½ÑÑÑÑÐºÑÐ¸Ð¹ ÑÐ¸ÑÑÐµÐ¼Ñ Cas12d1 Ñ ÐºÐ°ÑÐ°Ð»Ð¸ÑÐ¸ÑÐµÑÐºÐ¸ Ð½ÐµÐ°ÐºÑÐ¸Ð²Ð½ÑÐ¼ Ð½ÑÐºÐ»ÐµÐ°Ð·Ð½ÑÐ¼ Ð´Ð¾Ð¼ÐµÐ½Ð¾Ð¼; 3.Ð¢ÐµÑÑÐ¸ÑÐ¾Ð²Ð°Ð½Ð¸Ðµ Ð±ÐµÐ»ÐºÐ¾Ð² ÑÐ¸ÑÑÐµÐ¼Ñ Cas12d1 Ñ ÐºÐ°ÑÐ°Ð»Ð¸ÑÐ¸ÑÐµÑÐºÐ¸ Ð½ÐµÐ°ÐºÑÐ¸Ð²Ð½ÑÐ¼ Ð½ÑÐºÐ»ÐµÐ°Ð·Ð½ÑÐ¼ Ð´Ð¾Ð¼ÐµÐ½Ð¾Ð¼ in vitro. Ð Ð°Ð±Ð¾ÑÐ° Ð±ÑÐ»Ð° Ð²ÑÐ¿Ð¾Ð»Ð½ÐµÐ½Ð° Ð² ÐÐÐ Â«ÐÐ°Ð½Ð¾Ð±Ð¸Ð¾Â». Ð ÑÐµÐ·ÑÐ»ÑÑÐ°ÑÐµ Ð¿ÑÐ¾Ð´ÐµÐ»Ð°Ð½Ð½Ð¾Ð¹ ÑÐ°Ð±Ð¾ÑÑ Ð±ÑÐ»Ð¾ Ð¿Ð¾ÐºÐ°Ð·Ð°Ð½Ð¾, ÑÑÐ¾ ÑÐ¾ÑÐµÑÐ½ÑÐµ Ð¼Ð¸ÑÑÐµÐ½Ñ-Ð¼ÑÑÐ°ÑÐ¸Ð¸ D827A Ð¸ E913A Ð² Ð½ÑÐºÐ»ÐµÐ°Ð·Ð½Ð¾Ð¼ Ð´Ð¾Ð¼ÐµÐ½Ðµ RuvC Ð¿ÑÐ¸Ð²Ð¾Ð´ÑÑ Ðº Ð¾Ð±ÑÐ°Ð·Ð¾Ð²Ð°Ð½Ð¸Ñ Ð¼ÑÑÐ°Ð½ÑÐ½ÑÑ Ð±ÐµÐ»ÐºÐ¾Ð² dCasY1 D827A Ð¸ dCasY1 E913A, ÑÐ¿Ð¾ÑÐ¾Ð±Ð½ÑÑ ÑÐ²ÑÐ·ÑÐ²Ð°ÑÑÑÑ Ñ ÐÐÐ-Ð¼Ð¸ÑÐµÐ½ÑÑ, Ð¾Ð´Ð½Ð°ÐºÐ¾ Ð½Ðµ ÑÐ°Ð·ÑÐµÐ·Ð°ÑÑÐ¸Ðµ ÐµÐµ in vitro.
description: This work is devoted to the preparation and characterization of catalytically inactive effectors of the CRISPR/Cas12d1 system by introducing a missense mutation into the functional nuclease domain RuvC. Objective: to obtain and characterize catalytically inactive effectors of the CRISPR/Cas12d1 (CasY1) system. Tasks that were solved during the study: 1. The product of the alignment of the genetic sequences of the Cas12d1 system by the known sequences Cas12a and Cas12e. Determination of the functional domain of the Cas12d1 system and potential mutations that can lead to the loss of its catalytic activity; 2.Modeling and assembly of genetic constructs of the Cas12d1 system with a catalytically inactive nuclease domain; 3. Testing of Cas12d1 proteins with a catalytically inactive nuclease domain in vitro. The work was carried out in the Scientific research complex "Nanobio". As a result of the work done, it was shown that point missense mutations D827A and E913A in the RuvC nuclease domain lead to the formation of mutant proteins dCasY1 D827A and dCasY1 E913A, capable of binding to the target DNA, but not cutting it in vitro.