CRISPR-Cas9-based Genome Engineering to Generate Jurkat Reporter Models for HIV-1 Infection with Selected Proviral Integration Sites

Summary

We present a genome engineering workflow for the generation of new in vitro models for HIV-1 infection that recapitulate proviral integration at selected genomic sites. Targeting of HIV-derived reporters is facilitated by CRISPR-Cas9-mediated, site-specific genome manipulation. Detailed protocols for single-cell clone generation, screening, and correct targeting verification are provided.

Abstract

Human immunodeficiency virus (HIV) integrates its proviral DNA non-randomly into the host cell genome at recurrent sites and genomic hotspots. Here we present a detailed protocol for the generation of novel in vitro models for HIV infection with chosen genomic integration sites using CRISPR-Cas9-based genome engineering technology. With this method, a reporter sequence of choice can be integrated into a targeted, chosen genomic locus, reflecting clinically relevant integration sites.

In the protocol, the design of an HIV-derived reporter and choosing of a target site and gRNA sequence are described. A targeting vector with homology arms is constructed and transfected into Jurkat T cells. The reporter sequence is targeted to the selected genomic site by homologous recombination facilitated by a Cas9-mediated double-strand break at the target site. Single-cell clones are generated and screened for targeting events by flow cytometry and PCR. Selected clones are then expanded, and correct targeting is verified by PCR, sequencing, and Southern blotting. Potential off-target events of CRISPR-Cas9-mediated genome engineering are analyzed.

By using this protocol, novel cell culture systems that model HIV infection at clinically relevant integration sites can be generated. Although the generation of single-cell clones and verification of correct reporter sequence integration is time-consuming, the resulting clonal lines are powerful tools to functionally analyze proviral integration site choice.

Introduction

Integration of proviral DNA into the host genome upon infection is a critical step in the life cycle of human immunodeficiency virus (HIV). Following integration, HIV persists by establishing latency in long-lived CD4+ T cell subsets such as memory CD4+ T cells. HIV integration appears to be non-random^1,2. A number of genomic hotspots with recurrently integrated proviral DNA has been detected in several studies through the sequencing of integration sites in acutely and chronically infected individuals^2,3,4,....

Protocol

1. Targeting Strategy for Genome Engineering and Targeting Vector (tv) Design

NOTE: The first step of genome engineering involves selection and generation of the necessary tools for CRISPR-Cas9-mediated targeting. Selection of a genomic integration site locus, choice of cell type for targeting, and design of an HIV-derived reporter for integration should precede this step. This protocol describes targeting of an HIV-LTR_tdTomato_BGH-PA minimal reporter into Jurkat target cells. A flow chart of the workflow for CRISPR-Cas9-based targeting, generation, screening and verification of clonal lines is depicted in

Results

In this representative experiment we have chosen to target a minimal HIV-1-derived reporter consisting of a LTR, tdTomato-coding sequence, and polyA-signal sequence to two loci in intron 5 of the BACH2 gene¹⁷. The loci for targeting were chosen according to proximity to published recurrent integration sites found in different studies on primary T cells from HIV-infected patients^2,4,

Discussion

Here, we describe a protocol to generate HIV-1-derived Jurkat reporter models with chosen proviral integration sites applying CRISPR-Cas9-based genome engineering.

Several points of the protocol require careful attention during the planning stage. First, the locus to be targeted should be chosen carefully, as some loci might be easier to target than others (e.g., depending on the chromatin status of the region and the target sequence itself). Repetitive sequences are hard to clone int.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
pX330-U6-Chimeric_BB-cBh-hSpCas9	Addgene	42230	vector for expression of SpCas9 and gRNA
pMK	GeneArt		mammalian expression vector for cloning
cDNA3.1	Invitrogen	V79020	mammalian expression vector for cloning
BbsI	New England Biolabs	R0539S	restriction enzyme
NEBuilder Hifi DNA Assembly Cloning Kit	New England Biolabs	E5520S	Assembly cloning kit used for target vector generation
TaqPlus Precision PCR System	Agilent Technologies	600210	DNA polymerase with proofreading activity used for amplification of homology arms (step 1.2.2.2), verification of integration site and reporter sequence (step 3.3.3 and 3.3.5), generation of genomic probe for Southern blot (step 3.4.1.5) and analysis of off-target events (step 3.5.4)
96-well tissue culture plate (round-bottom)	TPP	92097	tissue culture plates for dilution plating
Phusion High-Fidelity DNA polymerase	New England Biolabs	M0530 L	DNA polymerase used for detection of targeting events (step 2.4.2) and generation ofreporter-specific probe for Southern blot (step 3.4.1.4)
Dimethyl sulfoxide (DMSO)	Sigma-Aldrich	D9170	dimethyl sulfoxide as PCR additive
Magnesium Chloride (MgCl2) Solution	New England Biolabs	B9021S	MgCl2 solution as PCR additive
Deoxynucleotide (dNTP) Solution Mix	New England Biolabs	N0447S	dNTP mixture with 10 mM of each nt for PCR reactions
5PRIME HotMasterMix	5PRIME	2200400	ready-to-use PCR mix used for screening PCR (step 3.2.11)
QIAamp DNA blood mini kit	Qiagen	51106	DNA isolation and purification kit
QIAquick PCR Purification Kit	Qiagen	28106	PCR Purification Kit
RPMI 1640 without glutamine	Lonza	BE12-167F	cell culture medium
Fetal Bovine Serum South Africa Charge	PAN Biotech	P123002	cell culture medium supplement
L-glutamine	Biochrom	K 0282	cell culture medium supplement
Penicillin/Streptomycin 10.000 U/mL/ 10.000 µg/mL	Biochrom	A 2212	cell culture medium supplement
Gibco Opti-MEM Reduced Serum Media	Thermo Fisher Scientific	31985062	cell culture medium with reduced serum concentration optimized for transfection
TransIT-Jurkat	Mirus Bio	MIR2125	transfection reagent
phorbol 12-myristate 13-acetate	Sigma-Aldrich	P8139-1MG	cell culture reagent
Ionomycin	Sigma-Aldrich	I0634-1MG	cell culture reagent
Syringe-driven filter unit, PES membrane, 0,22 µm	Millex	SLGP033RB	filter unit for sterile filtration
Heracell 150i incubator	Thermo Fisher Scientific	51026280	tissue culture incubator
Amershan Hybond-N+	GE Healthcare	RPN1520B	positively charged nylon membrane for DNA and RNA blotting
Stratalinker 1800	Stratagene	400072	UV crosslinker
High Prime	Roche	11585592001	kit for labeling of DNA with radioactive dCTP using random oligonucleotides as primers
illustra ProbeQuant G-50 Micro Columns	GE Healthcare	28-9034-08	chromatography spin-columns for purification of labeled DNA