TY - JOUR

T1 - Development of the Cas12a-based microdeletion and microinsertion detection system

AU - Чиринскайте, Ангелина Валерьевна

AU - Сопова, Юлия Викторовна

AU - Леонова, Елена Ивановна

AU - Зелинский, Андрей

PY - 2023/12/4

Y1 - 2023/12/4

N2 - CRISPR/Cas-based systems are widely used as genome editing systems, nucleic acid detection systems and molecular visualization instruments [1]. In our laboratory using CRISPR/Cas9 technology we have obtained several KO mouse lines harboring deletions ranging from 2 to 20 base pairs. While 20 bp deletions are easily PCR-detected, when it comes to 2 bp deletions it is essential to genotype numerous mice using time-consuming Sanger sequencing. We propose a microdeletion/microinsertion detection system based on Cas12a nuclease from Lachnospiraceae bacterium (LbCas12a). Its active complex consists of the Cas12a enzyme and one crisprRNA [2]. A special sequence called protospacer adjacent motif (PAM) is required for target recognition by LbCas12a. In our laboratory we have discovered new PAM TTAA recognized by LbCas12a [3]. Via agarose electrophoresis and fluorescent analysis using FAM-labeled probes we have shown that new PAM allowed detection of 1 bp substitutions in target DNA in vitro. Also we have tested different FAM-labeled probes and have shown that AT-rich probes longer than 10 bp are cleaved most effectively. Finally we have used our system for detecting 2 bp deletions in Pde6b-KO mice and Grin3A-KO mice and successfully distinguished these mice from wild type mice.

AB - CRISPR/Cas-based systems are widely used as genome editing systems, nucleic acid detection systems and molecular visualization instruments [1]. In our laboratory using CRISPR/Cas9 technology we have obtained several KO mouse lines harboring deletions ranging from 2 to 20 base pairs. While 20 bp deletions are easily PCR-detected, when it comes to 2 bp deletions it is essential to genotype numerous mice using time-consuming Sanger sequencing. We propose a microdeletion/microinsertion detection system based on Cas12a nuclease from Lachnospiraceae bacterium (LbCas12a). Its active complex consists of the Cas12a enzyme and one crisprRNA [2]. A special sequence called protospacer adjacent motif (PAM) is required for target recognition by LbCas12a. In our laboratory we have discovered new PAM TTAA recognized by LbCas12a [3]. Via agarose electrophoresis and fluorescent analysis using FAM-labeled probes we have shown that new PAM allowed detection of 1 bp substitutions in target DNA in vitro. Also we have tested different FAM-labeled probes and have shown that AT-rich probes longer than 10 bp are cleaved most effectively. Finally we have used our system for detecting 2 bp deletions in Pde6b-KO mice and Grin3A-KO mice and successfully distinguished these mice from wild type mice.

KW - CRISPR/Cas

KW - LbCas12a

KW - mice

UR - https://www.mendeley.com/catalogue/101293a1-7620-3162-80f3-c3fdcce9e00f/

U2 - 10.17816/ecogen568454

DO - 10.17816/ecogen568454

M3 - Meeting Abstract

VL - 21

SP - 20

EP - 21

JO - ЭКОЛОГИЧЕСКАЯ ГЕНЕТИКА

JF - ЭКОЛОГИЧЕСКАЯ ГЕНЕТИКА

SN - 1811-0932

IS - спецвыпуск

Y2 - 3 October 2023 through 5 October 2023

ER -