title: Ð Ð¾Ð»Ñ Ð½ÑÐºÐ»ÐµÐ°Ð·Ð½Ð¾Ð³Ð¾ Ð¸ Ñ ÐµÐ»Ð¸ÐºÐ°Ð·Ð½Ð¾Ð³Ð¾ Ð´Ð¾Ð¼ÐµÐ½Ð¾Ð² Cas3 Ð² Ð¸Ð½ÑÐµÑÑÐµÑÐµÐ½ÑÐ¸Ð¸ Ð¸ Ð¿ÑÐ°Ð¹Ð¼Ð¸ÑÐ¾Ð²Ð°Ð½Ð½Ð¾Ð¹ Ð°Ð´Ð°Ð¿ÑÐ°ÑÐ¸Ð¸ Ð² ÑÐ¸ÑÑÐµÐ¼Ðµ CRISPR-Cas I-E
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: The role of the Cas3 nuclease and helicase domains in interference and primed adaptation in the type I-E CRISPR-Cas system
Creators: name: ÐÐ°ÑÐ»Ð¸Ð½, ÐÐ¸ÐºÐ¸ÑÐ°
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: 2022
Subjects: CRISPR-Cas
Ð½ÑÐºÐ»ÐµÐ°Ð·Ð°-Ñ ÐµÐ»Ð¸ÐºÐ°Ð·Ð° Cas3
RecBCD
Ð¿ÑÐ°Ð¹Ð¼Ð¸ÑÐ¾Ð²Ð°Ð½Ð½Ð°Ñ Ð°Ð´Ð°Ð¿ÑÐ°ÑÐ¸Ñ
Ð¸Ð½ÑÐµÑÑÐµÑÐµÐ½ÑÐ¸Ñ
nuclease-helicase Cas3
primed adaptation
interference

description: Ð¢ÐµÐ¼Ð° Ð²ÑÐ¿ÑÑÐºÐ½Ð¾Ð¹ ÐºÐ²Ð°Ð»Ð¸ÑÐ¸ÐºÐ°ÑÐ¸Ð¾Ð½Ð½Ð¾Ð¹ ÑÐ°Ð±Ð¾ÑÑ: Â«Ð Ð¾Ð»Ñ Ð½ÑÐºÐ»ÐµÐ°Ð·Ð½Ð¾Ð³Ð¾ Ð¸ ÑÐµÐ»Ð¸ÐºÐ°Ð·Ð½Ð¾Ð³Ð¾ Ð´Ð¾Ð¼ÐµÐ½Ð¾Ð² Cas3 Ð² Ð¸Ð½ÑÐµÑÑÐµÑÐµÐ½ÑÐ¸Ð¸ Ð¸ Ð¿ÑÐ°Ð¹Ð¼Ð¸ÑÐ¾Ð²Ð°Ð½Ð½Ð¾Ð¹ Ð°Ð´Ð°Ð¿ÑÐ°ÑÐ¸Ð¸ Ð² ÑÐ¸ÑÑÐµÐ¼Ðµ CRISPR-Cas I-EÂ».Ð¦ÐµÐ»ÑÑ Ð´Ð°Ð½Ð½Ð¾Ð¹ ÑÐ°Ð±Ð¾ÑÑ ÑÐ²Ð»ÑÐµÑÑÑ Ð¾Ð¿ÑÐµÐ´ÐµÐ»ÐµÐ½Ð¸Ðµ ÑÐ¾Ð»Ð¸ ÐºÐ»ÑÑÐµÐ²ÑÑ Ð°Ð¼Ð¸Ð½Ð¾ÐºÐ¸ÑÐ»Ð¾ÑÐ½ÑÑ Ð¾ÑÑÐ°ÑÐºÐ¾Ð² ÑÐµÐ»Ð¸ÐºÐ°Ð·Ð½Ð¾Ð³Ð¾ Ð¸ Ð½ÑÐºÐ»ÐµÐ°Ð·Ð½Ð¾Ð³Ð¾ Ð´Ð¾Ð¼ÐµÐ½Ð¾Ð² Cas3, Ð° ÑÐ°ÐºÐ¶Ðµ ÑÐ¾Ð»Ñ Ð½ÑÐºÐ»ÐµÐ°Ð·Ñ RecBCD Ð² Ð¿ÑÐ°Ð¹Ð¼Ð¸ÑÐ¾Ð²Ð°Ð½Ð½Ð¾Ð¹ Ð°Ð´Ð°Ð¿ÑÐ°ÑÐ¸Ð¸ Ð¸ Ð¸Ð½ÑÐµÑÑÐµÑÐµÐ½ÑÐ¸Ð¸ Ð² ÑÐ¸ÑÑÐµÐ¼Ðµ CRISPR-Cas I-EÂ Escherichia coli. Ð Ð¸ÑÑÐ»ÐµÐ´Ð¾Ð²Ð°Ð½Ð¸Ð¸ Ð¸ÑÐ¿Ð¾Ð»ÑÐ·Ð¾Ð²Ð°Ð½ ÑÑÐ°Ð¼Ð¼Â E. coliÂ Ñ Ð°Ð²ÑÐ¾ÑÐ°ÑÐ³ÐµÑÐ¸ÑÑÑÑÐµÐ¹ ÑÐ¸ÑÑÐµÐ¼Ð¾Ð¹ CRISPR-Cas, ÐºÐ¾ÑÐ¾ÑÐ°Ñ ÑÐ¾Ð´ÐµÑÐ¶Ð¸Ñ CRISPR-ÐºÐ°ÑÑÐµÑÑ Ñ Ð¾Ð´Ð½Ð¸Ð¼ ÑÐ¿ÐµÐ¹ÑÐµÑÐ¾Ð¼, Ð¼Ð¸ÑÐµÐ½ÑÑ ÐºÐ¾ÑÐ¾ÑÐ¾Ð³Ð¾ ÑÐ²Ð»ÑÐµÑÑÑ Ð¿ÑÐ¾ÑÐ¾ÑÐ¿ÐµÐ¹ÑÐµÑ Ð² ÑÐ¾Ð±ÑÑÐ²ÐµÐ½Ð½Ð¾Ð¼ Ð³ÐµÐ½Ð¾Ð¼Ðµ ÐºÐ»ÐµÑÐºÐ¸.ÐÐ¾ÐºÐ°Ð·Ð°Ð½Ð¾, ÑÑÐ¾ Ð²Ð½ÐµÑÐµÐ½Ð¸Ðµ Ð°Ð¼Ð¸Ð½Ð¾ÐºÐ¸ÑÐ»Ð¾ÑÐ½ÑÑ Ð·Ð°Ð¼ÐµÐ½ Ð² Ð½ÑÐºÐ»ÐµÐ°Ð·Ð½ÑÐ¹ (H74A, H74A Ð¸ D75A) Ð¸ ÑÐµÐ»Ð¸ÐºÐ°Ð·Ð½ÑÐ¹ (D452A, K320N) Ð´Ð¾Ð¼ÐµÐ½Ñ Cas3 ÑÐ½Ð¸Ð¶Ð°ÐµÑ ÑÑÑÐµÐºÑÐ¸Ð²Ð½Ð¾ÑÑÑ Ð¸Ð½ÑÐµÑÑÐµÑÐµÐ½ÑÐ¸Ð¸. Ð¡ Ð¿Ð¾Ð¼Ð¾ÑÑÑ Ð²ÑÑÐ¾ÐºÐ¾Ð¿ÑÐ¾Ð¸Ð·Ð²Ð¾Ð´Ð¸ÑÐµÐ»ÑÐ½Ð¾Ð³Ð¾ ÑÐµÐºÐ²ÐµÐ½Ð¸ÑÐ¾Ð²Ð°Ð½Ð¸Ñ Ð¿Ð¾ÐºÐ°Ð·Ð°Ð½Ð¾, ÑÑÐ¾ Ð²ÑÐµ Ð¸Ð·ÑÑÐµÐ½Ð½ÑÐµ Ð·Ð°Ð¼ÐµÐ½Ñ Ð¾ÐºÐ°Ð·ÑÐ²Ð°ÑÑ Ð²Ð»Ð¸ÑÐ½Ð¸Ðµ Ð½Ð° ÑÑÑÐµÐºÑÐ¸Ð²Ð½Ð¾ÑÑÑ Ð¿ÑÐ°Ð¹Ð¼Ð¸ÑÐ¾Ð²Ð°Ð½Ð½Ð¾Ð¹ Ð°Ð´Ð°Ð¿ÑÐ°ÑÐ¸Ð¸, Ð½Ð¾ Ð² ÑÐ°Ð·Ð½Ð¾Ð¹ ÑÑÐµÐ¿ÐµÐ½Ð¸. ÐÐ°Ð¼ÐµÐ½Ð° H74A Ð² Ð½ÑÐºÐ»ÐµÐ°Ð·Ð½Ð¾Ð¼ Ð´Ð¾Ð¼ÐµÐ½Ðµ ÑÐ½Ð¸Ð¶Ð°ÐµÑ ÑÑÑÐµÐºÑÐ¸Ð²Ð½Ð¾ÑÑÑ Ð¿ÑÐ°Ð¹Ð¼Ð¸ÑÐ¾Ð²Ð°Ð½Ð½Ð¾Ð¹ Ð°Ð´Ð°Ð¿ÑÐ°ÑÐ¸Ð¸ Ð² 50 ÑÐ°Ð·. ÐÑÑÐ°ÑÐ¾ÑÐ½ÑÐ¹ ÑÑÐ¾Ð²ÐµÐ½Ñ Ð¿ÑÐ°Ð¹Ð¼Ð¸ÑÐ¾Ð²Ð°Ð½Ð½Ð¾Ð¹ Ð°Ð´Ð°Ð¿ÑÐ°ÑÐ¸Ð¸ Ð² ÐºÐ»ÐµÑÐºÐ°Ñ, ÑÐ¾Ð´ÐµÑÐ¶Ð°ÑÐ¸Ñ Cas3 H74A , Ð¾Ð±ÑÑÐ»Ð¾Ð²Ð»ÐµÐ½ Ð½ÐµÐ¿Ð¾Ð»Ð½Ð¾Ð¹ Ð¸Ð½Ð°ÐºÑÐ¸Ð²Ð°ÑÐ¸ÐµÐ¹ Ð½ÑÐºÐ»ÐµÐ°Ð·Ð½Ð¾Ð¹ Ð°ÐºÑÐ¸Ð²Ð½Ð¾ÑÑÐ¸ Cas3. ÐÐ¼Ð¸Ð½Ð¾ÐºÐ¸ÑÐ»Ð¾ÑÐ½Ð°Ñ Ð·Ð°Ð¼ÐµÐ½Ð° D452A Ð¿ÑÐ¸Ð²Ð¾Ð´Ð¸Ñ Ðº ÑÑÑÑÑÐµÐºÑÐ°ÑÐ½Ð¾Ð¼Ñ ÑÐ½Ð¸Ð¶ÐµÐ½Ð¸Ñ ÑÑÑÐµÐºÑÐ¸Ð²Ð½Ð¾ÑÑÐ¸ Ð°Ð´Ð°Ð¿ÑÐ°ÑÐ¸Ð¸. ÐÐ°Ð¼ÐµÐ½Ð° K320N Ð² ~ 2 ÑÐ°Ð·Ð° ÑÐ½Ð¸Ð¶Ð°ÐµÑ ÑÑÑÐµÐºÑÐ¸Ð²Ð½Ð¾ÑÑÑ Ð¿ÑÐ°Ð¹Ð¼Ð¸ÑÐ¾Ð²Ð°Ð½Ð½Ð¾Ð¹ Ð°Ð´Ð°Ð¿ÑÐ°ÑÐ¸Ð¸ Ð² PAM-Ð¿ÑÐ¾ÐºÑÐ¸Ð¼Ð°Ð»ÑÐ½Ð¾Ð¹ Ð¾Ð±Ð»Ð°ÑÑÐ¸ Ð¿ÑÐ¾ÑÐ¾ÑÐ¿ÐµÐ¹ÑÐµÑÐ°-Ð¼Ð¸ÑÐµÐ½Ð¸ Ð¸ Ð² ~ 200 ÑÐ°Ð· Ð² PAM-Ð´Ð¸ÑÑÐ°Ð»ÑÐ½Ð¾Ð¹ Ð¾Ð±Ð»Ð°ÑÑÐ¸. ÐÐ½Ð³Ð¸Ð±Ð¸ÑÐ¾Ð²Ð°Ð½Ð¸Ðµ Ð¿ÑÐ°Ð¹Ð¼Ð¸ÑÐ¾Ð²Ð°Ð½Ð½Ð¾Ð¹ Ð°Ð´Ð°Ð¿ÑÐ°ÑÐ¸Ð¸ Ð² PAM-Ð´Ð¸ÑÑÐ°Ð»ÑÐ½Ð¾Ð¹ Ð¾Ð±Ð»Ð°ÑÑÐ¸ Ð² ÑÑÐ°Ð¼Ð¼Ðµ Ñ Cas3 K320N Ð¾Ð±ÑÑÐ»Ð¾Ð²Ð»ÐµÐ½Ð¾ ÑÐµÐ»Ð¸ÐºÐ°Ð·Ð¾Ð¹-Ð½ÑÐºÐ»ÐµÐ°Ð·Ð¾Ð¹ RecBCD.
description: The subject of the graduate qualification work is Â«The role of the Cas3 nuclease and helicase domains in interference and primed adaptation in the type I-E CRISPR-Cas systemÂ».The given work is devoted to elucidating the role of key amino acid residues of the Cas3 helicase and nuclease domains, as well as the role of the RecBCD nuclease in primed adaptation and interference in the type I-E CRISPR-Cas system ofÂ Escherichia coli. We used anÂ E. coliÂ strain with the CRISPR-Cas self-targeting system, which contains a CRISPR array with a single spacer targeting a chromosomal protospacer.We demonstrate that amino acid substitutions in the nuclease (H74A, H74A, and D75A) and helicase (D452A, K320N) domains of Cas3 reduce the interference efficiency. Using high-throughput sequencing, we show that all of the studied substitutions affect the primed adaptation efficiency, but to a different degree. The H74A substitution in the nuclease domain reduces the primed adaptation efficiency by 50 times. The residual level of primed adaptation in cells containing Cas3 H74A is due to incomplete inactivation of the Cas3 nuclease activity. The D452A substitution leads to a thousandfold decrease in the adaptation efficiency. The K320N substitution reduces the primed adaptation efficiency by ~2 times in the PAM-proximal region of the priming protospacer and by ~200 times in the PAM-distal region. The inhibition of primed adaptation in the PAM-distal region in the strain with Cas3 K320N is due to the RecBCD helicase-nuclease.