Expansion and Precise CRISPR-Cas9 Gene Repair of Autologous T-Memory Stem Cells from Patients with T-Cell Immunodeficiencies.

The adoptive transfer of autologous, long-lived, gene-repaired T cells is a promising way to treat inherited T-cell immunodeficiencies. However, adoptive T-cell therapies require a large number of T cells to be manipulated and infused back into the patient. This poses a challenge in primary immunodeficiencies that manifest early in childhood and where only small volumes of blood samples may be available.

In vivo CRISPR/Cas9-mediated screen reveals a critical function of TFDP1 and E2F4 transcription factors in hematopoiesis.

Hematopoiesis is a continuous process of blood cell production driven by hematopoietic stem and progenitor cells (HSPCs) in the bone marrow. Proliferation and differentiation of HSPCs are regulated by complex transcriptional networks. In order to identify transcription factors with key roles in HSPC-mediated hematopoietic reconstitution, we developed an efficient and robust CRISPR/Cas9-based in vivo genetic screen.

Precise CRISPR-Cas9 gene repair in autologous memory T cells to treat familial hemophagocytic lymphohistiocytosis.

Familial hemophagocytic lymphohistiocytosis (FHL) is an inherited, often fatal immune deficiency characterized by severe systemic hyperinflammation. Although allogeneic bone marrow transplantation can be curative, more effective therapies are urgently needed. FHL is caused by inactivating mutations in proteins that regulate cellular immunity.

Application of a Spacer-nick Gene-targeting Approach to Repair Disease-causing Mutations with Increased Safety.

The CRISPR/Cas9 system is a powerful tool for gene repair that holds great potential for gene therapy to cure monogenic diseases. Despite intensive improvement, the safety of this system remains a major clinical concern. In contrast to Cas9 nuclease, Cas9 nickases with a pair of short-distance (38-68 bp) PAM-out single-guide RNAs (sgRNAs) preserve gene repair efficiency while strongly reducing off-target effects.

A CRISPR/Cas9-mediated screen identifies determinants of early plasma cell differentiation.

Introduction: The differentiation of B cells into antibody-secreting plasma cells depends on cell division-coupled, epigenetic and other cellular processes that are incompletely understood.

Methods: We have developed a CRISPR/Cas9-based screen that models an early stage of T cell-dependent plasma cell differentiation and measures B cell survival or proliferation versus the formation of CD138+ plasmablasts. Here, we refined and extended this screen to more than 500 candidate genes that are highly expressed in plasma cells.

Efficient CRISPR-Cas9-mediated mutagenesis in primary human B cells for identifying plasma cell regulators.

Human B lymphocytes are attractive targets for immunotherapies in autoantibody-mediated diseases. Gene editing technologies could provide a powerful tool to determine gene regulatory networks regulating B cell differentiation into plasma cells, and identify novel therapeutic targets for prevention and treatment of autoimmune disorders. Here, we describe a new approach that uses CRISPR-Cas9 technology to target genes in primary human B cells for identifying plasma cell regulators.

Precise CRISPR-Cas-mediated gene repair with minimal off-target and unintended on-target mutations in human hematopoietic stem cells.

While CRISPR-Cas9 is key for the development of gene therapy, its potential off-target mutations are still a major concern. Here, we establish a "spacer-nick" gene correction approach that combines the Cas9 nickase with a pair of PAM-out sgRNAs at a distance of 200 to 350 bp. In combination with adeno-associated virus (AAV) serotype 6 template delivery, our approach led to efficient HDR in human hematopoietic stem and progenitor cells (HSPCs including long-term HSCs) and T cells, with minimal NHEJ-mediated on-target mutations.

CRISPR-Cas9-Mediated ELANE Mutation Correction in Hematopoietic Stem and Progenitor Cells to Treat Severe Congenital Neutropenia.

Severe congenital neutropenia (SCN) is a monogenic disorder. SCN patients are prone to recurrent life-threatening infections. The main causes of SCN are autosomal dominant mutations in the ELANE gene that lead to a block in neutrophil differentiation.

Protocol for Efficient CRISPR/Cas9/AAV-Mediated Homologous Recombination in Mouse Hematopoietic Stem and Progenitor Cells.

Mutations that accumulate in self-renewing hematopoietic stem and progenitor cells (HSPCs) can cause severe blood disorders. To model such disorders in mice, we developed a CRISPR/Cas9/adeno-associated virus (AAV)-based system to knock in and repair genes by homologous recombination in mouse HSPCs. Here, we provide a step-by-step protocol to achieve high efficiency of gene knockin in mouse HSPCs, while maintaining engraftment capacity.

Functional interplay of Epstein-Barr virus oncoproteins in a mouse model of B cell lymphomagenesis.

Epstein-Barr virus (EBV) is a B cell transforming virus that causes B cell malignancies under conditions of immune suppression. EBV orchestrates B cell transformation through its latent membrane proteins (LMPs) and Epstein-Barr nuclear antigens (EBNAs). We here identify secondary mutations in mouse B cell lymphomas induced by LMP1, to predict and identify key functions of other EBV genes during transformation.

Corrigendum: Enhancement of Precise Gene Editing by the Association of Cas9 With Homologous Recombination Factors.

[This corrects the article DOI: 10.3389/fgene.2019.

Efficient CRISPR/Cas9-Mediated Gene Knockin in Mouse Hematopoietic Stem and Progenitor Cells.

Mutations accumulating in hematopoietic stem and progenitor cells (HSPCs) during development can cause severe hematological disorders. Modeling these mutations in mice is essential for understanding their functional consequences. Here, we describe an efficient CRISPR/Cas9-based system to knock in and repair genes in mouse HSPCs.

Efficient and Precise CRISPR/Cas9-Mediated MECP2 Modifications in Human-Induced Pluripotent Stem Cells.

Patients with Rett syndrome (RTT) have severe mental and physical disabilities. The majority of RTT patients carry a heterozygous mutation in methyl-CpG binding protein 2 (MECP2), an X-linked gene encoding an epigenetic factor crucial for normal nerve cell function. No curative therapy for RTT syndrome exists, and cellular mechanisms are incompletely understood.

Enhancement of Precise Gene Editing by the Association of Cas9 With Homologous Recombination Factors.

The CRISPR-Cas9 system is used for genome editing in mammalian cells by introducing double-strand breaks (DSBs) which are predominantly repaired via non-homologous end joining (NHEJ) or to lesser extent by homology-directed repair (HDR). To enhance HDR for improving the introduction of precise genetic modifications, we tested fusion proteins of Cas9 nuclease with HDR effectors to enforce their localization at DSBs. Using a traffic-light DSB repair reporter (TLR) system for the quantitative detection of HDR and NHEJ events in human HEK cells we found that Cas9 fusions with CtIP, Rad52, and Mre11, but not Rad51C promote HDR up to twofold in human cells and significantly reduce NHEJ events.

BCR-dependent lineage plasticity in mature B cells.

B2 cells engage in classical antibody responses, whereas B1 cells are considered carriers of innate immunity, biased toward recognizing epitopes present on the surfaces of common pathogens and self antigens. To explore the role of B cell antigen receptor (BCR) specificity in driving B1 cell differentiation, we developed a transgenic system allowing us to change BCR specificity in B cells in an inducible and programmed manner. Mature B2 cells differentiated into bona fide B1 cells upon acquisition of a B1 cell-typical self-reactive BCR through a phase of proliferative expansion.

sgRNA Sequence Motifs Blocking Efficient CRISPR/Cas9-Mediated Gene Editing.

Cas9 nucleases can be programmed with single guide RNAs (sgRNAs) to mediate gene editing. High CRISPR/Cas9-mediated gene knockout efficiencies are essential for genetic screens and critically depend on the properties of the sgRNAs used. The specificity of an sgRNA is defined by its targeting sequence.

A novel allele for inducible Cre expression in germinal center B cells.

The germinal center reaction is essential for efficient humoral immunity, but it can also give rise to B cell lymphomas. Cre/loxP-mediated conditional gene knock-out or knock-in can be used for the genetic manipulation of germinal center B cells in vivo. Here we present a novel allele, Cγ1-CreERT2, that allows for timed activation of Cre recombinase in a small fraction of germinal center B cells.

Nuclear FOXO1 promotes lymphomagenesis in germinal center B cells.

Forkhead box class O1 (FOXO1) acts as a tumor suppressor in solid tumors. The oncogenic phosphoinositide-3-kinase (PI3K) pathway suppresses FOXO1 transcriptional activity by enforcing its nuclear exclusion upon AKT-mediated phosphorylation. We show here abundant nuclear expression of FOXO1 in Burkitt lymphoma (BL), a germinal center (GC) B-cell-derived lymphoma whose pathogenesis is linked to PI3K activation.

CRISPR/Cas9-Mediated In Vitro Mutagenesis in GC-Like B Cells.

The CRISPR/Cas9 technology has developed into a powerful tool for genome editing, both in terms of gene silencing and the insertion of precise mutations. However, the application of CRISPR/Cas9-mediated mutagenesis in primary immune cells, in particular in B cells, is still in its infancy because of the difficulty to deliver the CRISPR/Cas9 system into these cells. Here, we describe a new method to use CRISPR/Cas9 for manipulating genes in germinal center (GC)-like B cells in vitro.

Efficient CRISPR-mediated mutagenesis in primary immune cells using CrispRGold and a C57BL/6 Cas9 transgenic mouse line.

Applying clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9)-mediated mutagenesis to primary mouse immune cells, we used high-fidelity single guide RNAs (sgRNAs) designed with an sgRNA design tool (CrispRGold) to target genes in primary B cells, T cells, and macrophages isolated from a Cas9 transgenic mouse line. Using this system, we achieved an average knockout efficiency of 80% in B cells. On this basis, we established a robust small-scale CRISPR-mediated screen in these cells and identified genes essential for B-cell activation and plasma cell differentiation.

Isolation of Eosinophils from the Lamina Propria of the Murine Small Intestine.

Only recently has it become apparent that eosinophils play a crucial role in mucosal immune homeostasis. Although eosinophils are the main cellular component of the lamina propria of the gastrointestinal tract, they have often been overlooked because they express numerous markers, which are normally used to characterize macrophages and/or dendritic cells. To study their function in mucosal immunity, it is important to isolate them with high purity and viability.

Efficient generation of Rosa26 knock-in mice using CRISPR/Cas9 in C57BL/6 zygotes.

Background: The CRISPR/Cas9 system is increasingly used for gene inactivation in mouse zygotes, but homology-directed mutagenesis and use of inbred embryos are less established. In particular, Rosa26 knock-in alleles for the insertion of transgenes in a genomic 'safe harbor' site, have not been produced. Here we applied CRISPR/Cas9 for the knock-in of 8-11 kb inserts into Rosa26 of C57BL/6 zygotes.

PI3 Kinase and FOXO1 Transcription Factor Activity Differentially Control B Cells in the Germinal Center Light and Dark Zones.

Phosphatidylinositol 3' OH kinase (PI3K) signaling and FOXO transcription factors play opposing roles at several B cell developmental stages. We show here abundant nuclear FOXO1 expression in the proliferative compartment of the germinal center (GC), its dark zone (DZ), and PI3K activity, downregulating FOXO1, in the light zone (LZ), where cells are selected for further differentiation. In the LZ, however, FOXO1 was expressed in a fraction of cells destined for DZ reentry.

Pop in, pop out: a novel gene-targeting strategy for use with CRISPR-Cas9.

The CRISPR-Cas9 system is frequently used to create small deletions in the genomes of mammalian cells, but the isolation of precisely targeted mutants is still challenging. A new, two-step 'pop in & out' targeting approach facilitates this task.

Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells.

The insertion of precise genetic modifications by genome editing tools such as CRISPR-Cas9 is limited by the relatively low efficiency of homology-directed repair (HDR) compared with the higher efficiency of the nonhomologous end-joining (NHEJ) pathway. To enhance HDR, enabling the insertion of precise genetic modifications, we suppressed the NHEJ key molecules KU70, KU80 or DNA ligase IV by gene silencing, the ligase IV inhibitor SCR7 or the coexpression of adenovirus 4 E1B55K and E4orf6 proteins in a 'traffic light' and other reporter systems. Suppression of KU70 and DNA ligase IV promotes the efficiency of HDR 4-5-fold.