Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells

Sakthi D. Moorthy; Jennifer A. Mitchell

doi:10.3791/53552

JoVE Journal > Developmental Biology

Biologia dello sviluppo

Generering CRISPR / Cas9 Mediated monoallelisk Sletninger til Studieinformationen Enhancer funktion i mus embryonale stamceller

Published: April 02, 2016

doi:

10.3791/53552

Sakthi D. Moorthy, Jennifer A. Mitchell

¹Department of Cell and Systems Biology,University of Toronto

Summary

Experimental validation of enhancer activity is best approached by loss-of-function analysis. Presented here is an efficient protocol that uses CRISPR/Cas9 mediated deletion to study allele-specific regulation of gene transcription in F1 ES cells which contain a hybrid genome (Mus musculus¹²⁹ x Mus castaneus).

Abstract

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often located distally from the regulated gene in intergenic regions or even within the body of another gene. The position independent nature of enhancer activity makes it difficult to match enhancers with the genes they regulate. Deletion of an enhancer region provides direct evidence for enhancer activity and is the gold standard to reveal an enhancer’s role in endogenous gene transcription. Conventional homologous recombination based deletion methods have been surpassed by recent advances in genome editing technology which enable rapid and precisely located changes to the genomes of numerous model organisms. CRISPR/Cas9 mediated genome editing can be used to manipulate the genome in many cell types and organisms rapidly and cost effectively, due to the ease with which Cas9 can be targeted to the genome by a guide RNA from a bespoke expression plasmid. Homozygous deletion of essential gene regulatory elements might lead to lethality or alter cellular phenotype whereas monoallelic deletion of transcriptional enhancers allows for the study of cis-regulation of gene expression without this confounding issue. Presented here is a protocol for CRISPR/Cas9 mediated deletion in F1 mouse embryonic stem (ES) cells (Mus musculus¹²⁹ x Mus castaneus). Monoallelic deletion, screening and expression analysis is facilitated by single nucleotide polymorphisms (SNP) between the two alleles which occur on average every 125 bp in these cells.

Introduction

Transkriptionelle regulatoriske elementer er afgørende for spatio-temporale finjustering af genekspression under udvikling ¹ og modifikation af disse elementer kan resultere i sygdom på grund af afvigende genekspression ^2. Mange sygdom-associerede områder identificeret ved genom bred forening undersøgelser er i ikke-kodende områder og har træk af transkriptionelle forstærkere ^3-4. Identificering enhancere og matche dem med de gener, de regulerer er kompliceret, da de ofte er placeret adskillige kilobaser væk fra generne, de regulerer og kan aktiveres i en vævsspecifik måde ^5-6. Enhancer forudsigelser er almindeligt baseret på histon modifikation mærker, mediator-cohesin komplekser og binding af celletype-specifikke transkriptionsfaktorer ^7-10. Validering af forudsagte enhancere er oftest sker gennem en vektor baseret assay, ved hvilken enhancer aktiverer ekspression af et reportergen ^11-12. Disse data giver valuable information om de lovgivningsmæssige potentiale formodede forstærkersekvenser men afslører ikke deres opgaver i deres endogene genomiske kontekst eller identificere de gener, de regulerer. Genom redigering tjener som et effektivt redskab til at studere funktionen af transkriptionelle regulatoriske elementer i deres endogene kontekst ved tab af funktion analyse.

Nylige fremskridt i genom redigering, nemlig CRISPR / Cas9 genom redigering system, lette efterforskningen af genom-funktion. Den CRISPR / Cas9 systemet er let at bruge og kan tilpasses til mange biologiske systemer. Den Cas9 protein er målrettet til et specifikt sted i genomet af en vejledning RNA (gRNA) ^13. Den SpCas9 / gRNA komplekset scanner genomet for sit mål genomiske sekvens, som skal være 5 'til en protospacer tilstødende motiv (PAM) sekvens, NGG ^14-15. Baseparring af gRNA til dens mål, en 20 nukleotid (nt) sekvens komplementær til gRNA, aktiverer SpCas9 nukleaseaktivitet resulterer i en dobbele streng brud (DSB) 3 bp opstrøms for PAM-sekvensen. Specificitet opnås gennem fuldstændig baseparring i gRNA frø region, 6-12 nt siden af PAM; omvendt ikke stemmer overens 5 'af frø sædvanligvis tolereres ^16-17. Den indførte DSB kan repareres enten af ikke-homolog ende sammenføjning (NHEJ) DNA-reparation eller homologi rettet reparation (HDR) mechanisms.NHEJ DNA-reparation skaber ofte insertion / deletion (indels) af et par bp på målstedet, der kan forstyrre den åbne læseramme (ORF) af et gen. For at generere større deletioner i genomet to gRNAs, som flankerer regionen af interesse, kan anvendes ^18-19. Denne fremgangsmåde er særligt anvendelig for studiet af transkriptionelle enhancere grupperet i locus kontrol regioner eller super-enhancere, der er større end konventionelle forstærkere ^9,18,20-22.

Monoallelisk sletninger er en værdifuld model til at studere cis -forordning af transskription. Den observerede Change i udskrift niveau efter monoallelisk sletning af en forstærker korrelerer til den rolle, som forstærker i genregulering uden de forstyrrende effekter, der kan opstå, når transkription af begge alleler påvirkes potentielt påvirke cellulære fitness. Evaluering reduceret ekspression er vanskeligt dog uden evnen til at skelne mellem den udgår af vildtype allelen. Endvidere genotypebestemmelse deletioner på hver allel uden evnen til at skelne mellem de to alleler er udfordrende, især for store deletioner af> 10 kb til 1 Mb ^23, i hvilken det er vanskeligt at amplificere hele vildtype region ved PCR. Anvendelsen af F1 ES celler frembragt ved krydsning Mus musculus ¹²⁹ med Mus castaneus tillader de to alleler, der skal differentieres ved allel-specifik PCR ^18,24. Den hybride genomet i disse celler letter allel-specifik deletion screening og ekspressionsanalyse. I gennemsnit er der en SNP hver 125 bp mellem disse to genomer, Hvilket giver fleksibilitet i primer design for ekspression og genotypebestemmelse analyser. Tilstedeværelsen af én SNP kan påvirke primer smeltetemperatur (T _m) og målrette specificitet i real-time kvantitativ PCR (qPCR) amplifikation tillader diskrimination af de to alleler ^25. Endvidere et mismatch i 3'-enden af primeren i høj grad påvirker evnen af DNA-polymerase til at forlænge fra primer forhindrer amplifikation af det uønskede allel target ^26. Beskrevet i det følgende protokol er anvendelsen af F1 ES celler til allelspecifikke enhancer deletioner af mere end 1 kb og efterfølgende ekspression analyse under anvendelse CRISPR / Cas9 genom redigering system (figur 1).

Figur 1. Enhancer sletning brug CRISPR / Cas9 at studere cis -regulation af genekspression. (A) F1 ES celler frembragt af en krydsning mellem Mus musculus ¹²⁹ og Mus castaneus anvendes til at give mulighed for allel specifik deletion. (B) To guide RNA'er (gRNA) anvendes til at inducere et stort Cas9-medieret deletion af enhancerregionen. (C) Primersæt bruges til at identificere store mono- og bi-allele deletioner. De orange primere er de indvendige primere, de lilla primere er de udvendige primere og de grønne primere de gRNA flankerende primere. (D) Ændringer i genekspression overvåges ved hjælp af allel-specifik qPCR. RFU betegner relative fluorescensenheder. Klik her for at se en større version af dette tal.

Protocol

1. Design og Konstruktion af gRNA For at slette transkriptionelle enhancerregioner bruge to gRNAs, en 5 'og en 3' af området af interesse. Brug musen UCSC genom browser spor genereret af Zhang laboratorium for at identificere unikke gRNA sekvenser (http://www.genome-engineering.org 15). Næste kontrollere disse gRNAs og deres tilstødende PAM for SNPs og indels bruger online værktøjer i Sanger Institute (www.sanger.ac.uk/sanger/Mouse_SnpViewer/rel-1211) 27-28. For at målrette…

Representative Results

Protokollen beskrevet her anvender F1 ES celler for at studere cis -regulering af genekspression i monoallelisk enhancer slettede celler dannet ved anvendelse CRISPR / Cas9 genom redigering (figur 1). Den gRNA og allel-specifikke primer design til genotypebestemmelse og genekspression er de vigtigste faktorer i denne fremgangsmåde. Hver allel-specifikke primer sæt skal valideres af qPCR at bekræfte allel specificitet. Allel-specifikke primere, som amplificere…

Discussion

CRISPR / Cas9 medieret genom redigering teknologi giver en enkel, hurtig og billig metode til genom modifikation. Metoden beskrevet her for at generere og analysere monoallelisk forstærker sletning til funktionel forstærker karakterisering udnytter SNPs i F1 museceller. Fordelene ved denne type fremgangsmåde er: 1) monoallelisk enhancer deletioner ikke producerer forstyrrende effekter, som opstår, når et kritisk enhancer udgår fra begge alleler, dvs. en stor reduktion i protein-niveauer i det regulerede g…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

We would like to thank all the members of the Mitchell lab for helpful discussions. This work was supported by the Canadian Institutes of Health Research, the Canada Foundation for Innovation and the Ontario Ministry of Research and Innovation (operating and infrastructure grants held by JAM).

Materials

Phusion High-Fidelity DNA Polymerase	NEB	M0530S	high fidelity DNA polymerase used in gRNA assembly
Gibson Assembly Master Mix	NEB	E2611L
gRNA_Cloning Vector	Addgene	41824	A target sequence is cloned into this vector to create the gRNA plasmid
pCas9_GFP	Addgene	44719	Codon-optimized SpCas9 and EGFP co-expression plasmid
AflII	NEB	R0520S
EcoRI	NEB	R3101S
Neon Transfection System 100 µL Kit	Life Technologies	MPK10096	Microporator transfection technology
prepGEM	ZyGEM	PT10500	genomic DNA extraction reagent
Nucleo Spin Gel & PCR Clean-up	Macherey-Nagel	740609.5
High-Speed Plasmid Mini Kit	Geneaid	PD300
Maxi Plasmid Kit Endotoxin Free	Geneaid	PME25
SYBR select mix for CFX	Life Technologies	4472942	qPCR reagent
iScript cDNA synthesis kit	Bio-rad	170-8891	Reverse transcription reagent
0.25% Trypsin with EDTA	Life Technologies	25200072
PBS without Ca/Mg²⁺	Sigma	D8537
0.5M EDTA	Bioshop	EDT111.500
HBSS	Life Technologies	14175095
1M HEPES	Life Technologies	13630080
BSA fraction V (7.5%)	Life Technologies	15260037
Max Efficiency DH5α competent cells	Invitrogen	18258012
FBS	ES cell qualified		FBS is subjected to a prior testing in mouse ES cells for pluripotency
DMSO	Sigma	D2650
Glutamax	Invitrogen	35050
DMEM	Life Technologies	11960069
Pencillin/Streptomycin	Invitrogen	15140
Sodium pyruvate	Invitrogen	11360
Non-essential aminoacid	Invitrogen	11140
β-mercaptoethanol	Sigma	M7522
96-well plate	Sarstedt	83.3924
Sealing tape	Sarstedt	95.1994
CoolCell LX	Biocision	BCS-405	alcohol-free cell freezing container
CHIR99021	Biovision	1748-5	Inhibitor for F1 ES cell culture
PD0325901	Invivogen	inh-pd32	Inhibitor for F1 ES cell culture
LIF	Chemicon	ESG1107	Inhibitor for F1 ES cell culture

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Moorthy, S. D., Mitchell, J. A. Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells. J. Vis. Exp. (110), e53552, doi:10.3791/53552 (2016).