Recombinant Macaca fascicularis C-C chemokine receptor type 5 (CCR5)

What is the biological significance of CCR5 in Macaca fascicularis?

CCR5 in Mauritian cynomolgus macaques (MCM, Macaca fascicularis) serves as a co-receptor for simian immunodeficiency virus (SIV) and HIV entry into cells, similar to its function in humans. The receptor is primarily expressed on leukocytes and plays pivotal roles in inflammatory responses and immune function . A 24-bp region in the macaque CCR5 gene has been identified as essential for its expression . Unlike humans, naturally occurring CCR5 mutations conferring HIV resistance are not documented in macaques, making them valuable models for studying genome editing approaches to HIV resistance .

How does Macaca fascicularis CCR5 compare structurally to human CCR5?

While the search results don't provide specific structural comparisons, research indicates functional homology between macaque and human CCR5. The essential regions for CCR5 expression in macaques have been identified, including a critical 24-bp region that researchers target when attempting to functionally delete CCR5 in these animals . This functional similarity makes macaques suitable models for studying HIV co-receptor interactions, despite potential structural differences. Researchers have successfully applied human CCR5-targeting strategies to macaque models, suggesting substantial conservation of critical functional domains .

What methods are used to isolate and express recombinant Macaca fascicularis CCR5?

Researchers typically isolate genomic DNA from macaque peripheral blood mononuclear cells using commercial kits such as Quick-DNA Miniprep (Zymo Research) . For recombinant expression, several approaches have been documented:

• PCR amplification of the CCR5 gene using primers flanking the coding region

• Cloning into expression vectors suited for mammalian cell expression

• Transfection into cell lines such as HEK293T for functional studies

For quality control, researchers verify CCR5 expression through:

• PCR amplification with primers that generate either short (613 bp) or long (2,925 bp) amplicons

• Gel electrophoresis to visualize amplicons (1.2%-1.5% agarose gels at 120V)

• DNA sequencing to confirm the integrity of the expressed gene

What CRISPR-Cas9 strategies have been successful for editing CCR5 in Macaca fascicularis?

Successful CRISPR-Cas9 editing of CCR5 in Mauritian cynomolgus macaques has been achieved using a dual-guide approach. Researchers design guide RNAs to encompass a 24-bp deletion essential for CCR5 expression in non-human primates . The specific methodology includes:

• Design of guide RNAs targeting conserved regions of the CCR5 gene

• Microinjection of CRISPR-Cas9 components (Cas9 protein and gRNAs) into one-cell stage embryos

• Cultivation of embryos to blastocyst stage

• PCR-based screening and whole genome sequencing to evaluate editing outcomes

Using this approach, researchers have achieved biallelic deletions in approximately 23-37% of MCM embryos . The dual-guide strategy targets two specific sites within the CCR5 gene to create a deletion that functionally inactivates the receptor, mimicking the protective effect seen with CCR5Δ32 in humans .

How can researchers verify on-target and off-target effects in CCR5-edited Macaca fascicularis models?

Comprehensive verification of CRISPR-Cas9 editing outcomes in CCR5-edited macaque models requires a multi-layered approach:

On-target verification:

• PCR amplification with primers flanking the target region (yielding 613 bp for wild-type and 415 bp for biallelic deletion mutants)

• Long-range PCR (2,925 bp amplicon) to detect large-scale deletions near the target site

• Gel electrophoresis to visualize amplicons

• Single-cell whole genome sequencing (WGS) to detect mosaicism and structural variants

Off-target analysis:

• Computational prediction of potential off-target sites using tools like Cas-OFFinder (allowing for three mismatches)

• WGS comparison between edited cells/embryos and parental DNA

• Sanger sequencing of PCR amplicons from predicted off-target regions

• Identification of de novo structural variants and single nucleotide variants not present in parental DNA

Research has revealed that PCR-based methods alone may underestimate the complexity of editing outcomes, as WGS has identified large-scale deletions (up to ~5.2 kb) and inversions that contribute to greater mosaicism than initially detected .

What is the frequency of mosaicism in CCR5-edited Macaca fascicularis embryos?

Mosaicism is a significant consideration in CRISPR-Cas9-edited macaque embryos. Single blastomere PCR analysis has revealed substantial mosaicism within individual embryos . Whole genome sequencing has further demonstrated that mosaicism is more prevalent than initially detected by PCR-based methods alone .

Specific findings on mosaicism include:

• Varied editing outcomes between blastomeres from the same embryo

• De novo structural variants unique to specific blastomeres

• Different large-scale deletions (ranging from the precise target deletion to ~5.2 kb deletions) in different cells of the same embryo

• Inversions detected in pairs of blastomeres within embryos

This mosaicism presents challenges for developing models with consistent genotypes and must be carefully considered when interpreting phenotypic results from genome-edited animals.

How does CCR5 editing in Macaca fascicularis compare to the human CCR5Δ32 mutation for HIV resistance?

The human CCR5Δ32 mutation confers resistance to HIV-1 infection, and transplantation of hematopoietic stem cells (HSCs) containing this mutation to HIV patients has led to HIV cure in some cases . Researchers aim to mimic this resistance through genetic editing of CCR5 in macaque models.

Key comparisons:

• Target region: While human CCR5Δ32 is a specific 32-bp deletion, macaque studies target a 24-bp region essential for CCR5 expression in non-human primates

• Efficiency: CRISPR-Cas9 editing has achieved biallelic deletions in ~23-37% of MCM embryos , whereas CCR5Δ32 occurs naturally in certain human populations

• Functional outcome: Both approaches aim to disrupt CCR5 function as an HIV co-receptor, though through different specific mutations

• Consistency: The human CCR5Δ32 is a defined mutation, while CRISPR-Cas9 editing can produce diverse outcomes, including large-scale deletions and mosaicism

The development of CCR5-edited macaque models provides a platform to determine mechanisms of HIV elimination following HSC transplantation and to develop clinical protocols for reproducible HIV cure .

What methodologies are used to evaluate SIV resistance in CCR5-modified Macaca fascicularis models?

Evaluation of SIV resistance in CCR5-modified macaques utilizes several methodological approaches:

• Viral challenge studies: Exposing edited animals to SIV and monitoring for infection

• Viral load monitoring: Quantitative measurement of plasma viral RNA

• Replication-competent viral reservoir (RCVR) analysis: Using statistical tools to calculate the size of the viral reservoir based on viral load and viral outgrowth data

• CD4+ T cell counts: Monitoring immune cell populations susceptible to SIV infection

• Antibody response analysis: Measuring the development of anti-SIV antibodies

• Time to viral rebound: After antiretroviral therapy interruption, measuring the time until viral loads become detectable again

Research has shown that CCR5-targeted treatments in macaques result in smaller RCVR sizes compared to control animals (p=0.01 and p=0.0002), demonstrating the effectiveness of these interventions .

What large-scale chromosomal anomalies have been observed in CRISPR-Cas9 editing of CCR5 in Macaca fascicularis?

Whole genome sequencing of CRISPR-Cas9-edited CCR5 in MCM embryos has revealed several types of large-scale chromosomal anomalies:

Additionally, homozygous insertions of 13 bp, 5 bp, 11 bp, and 33 bp were identified near one gRNA cut site in blastomere 5-4 .

These findings align with observations in other species, where CRISPR-Cas9 editing has resulted in unintended chromosomal anomalies, including large deletions and loss of heterozygosity. Such anomalies could potentially impact embryo viability and necessitate careful screening when developing edited animal models .

How can researchers optimize IVF and embryo handling protocols for CCR5 editing in Macaca fascicularis?

Successful genome editing in MCM embryos requires optimized protocols for ovarian stimulation, in vitro fertilization, and embryo culture. Key optimization strategies include:

• Ovarian stimulation protocol adaptation:

  • Modifications to hormonal regimens established for Chinese cynomolgus macaques

  • Tailoring protocols specifically for Mauritian cynomolgus macaques

• CRISPR-Cas9 microinjection timing:

  • Delivery of CRISPR-Cas9 components at the one-cell stage

  • Careful timing to minimize mosaicism while ensuring editing efficiency

• Embryo culture optimization:

  • Use of time-lapse imaging to assess embryonic developmental events in both control and microinjected embryos

  • Comparative analysis of developmental timing between edited and control embryos

• Quality control measures:

  • Single blastomere analysis to assess editing outcomes

  • Evaluation of embryo development rates

  • Screening for chromosomal anomalies that might affect embryo viability

These optimizations are essential for generating viable CCR5-edited embryos and ultimately establishing CCR5-mutant MCM models for HIV/SIV research.

What analytical approaches can detect subtle off-target effects in CCR5-edited Macaca fascicularis models?

Detecting subtle off-target effects requires sophisticated analytical approaches:

• Computational prediction and prioritization:

  • Use of tools like Cas-OFFinder allowing for varying numbers of mismatches (typically 3-5)

  • Prioritization of sites based on similarity to on-target sequence and genomic context

• Whole genome sequencing analysis:

  • Deep sequencing (>30x coverage for parental DNA)

  • Comparative analysis between edited samples and parental DNA

  • Detection of de novo mutations absent in parental genomes

• Single-cell sequencing approaches:

  • Individual blastomere analysis to detect mosaicism

  • Varying coverage analysis across chromosomes (from <1x to >70x) to detect structural variants

• Multi-caller validation:

  • Using multiple structural variant callers to confirm findings

  • Prioritizing variants called by at least two different callers

• PCR validation of predicted off-target sites:

  • Targeted amplification of predicted off-target regions

  • Sanger sequencing to confirm potential modifications

This multi-layered approach allows for comprehensive detection of both predicted and unexpected off-target effects that might impact model development and experimental interpretation.

How do CCR5-edited Macaca fascicularis models facilitate HIV cure strategies?

CCR5-edited macaque models provide critical insights for translational HIV cure research:

• Evaluating HSC transplantation strategies:

  • Modeling the Berlin patient and other cases where CCR5Δ32 HSC transplantation led to HIV cure

  • Determining mechanisms of HIV elimination following transplantation of allogeneic HSCs with CCR5 mutations

• Optimizing clinical protocols:

  • Development of reproducible HIV cure strategies

  • Evaluating factors contributing to successful versus unsuccessful outcomes

• Addressing physiological relevance:

  • Non-human primates are physiologically closer to humans than other laboratory models

  • MCMs specifically offer the advantage of high MHC allele sharing, making them valuable for stem cell therapy research

• Comparing intervention strategies:

  • Assessment of different CCR5-targeting approaches (gene editing, antibody-based interventions)

  • Comparative analysis of viral reservoir clearance between approaches

These models bridge the gap between basic HIV research and clinical applications, potentially accelerating the development of curative interventions for HIV infection.

What safety considerations must be addressed before clinical translation of CCR5 editing strategies from Macaca fascicularis models?

Research with CCR5-edited macaque models has identified several safety considerations crucial for clinical translation:

• Chromosomal instability:

  • CRISPR-Cas9 editing can result in large deletions (up to 22,471 bp) and inversions

  • Similar chromosomal anomalies have been observed in human embryos

• Off-target mutations:

  • Potential for unintended modifications at genomic sites with sequence similarity to the target

  • Long-term consequences of these modifications require monitoring

• Mosaicism:

  • Variable editing outcomes in different cells

  • Potential for unpredictable phenotypic outcomes

• Functional consequences:

  • CCR5 plays roles beyond HIV co-receptor function

  • Potential immunological impacts of CCR5 modification require evaluation

These findings suggest that further optimization of macaque embryo editing to avoid targeting errors would be essential for both model development and eventual clinical translation . Comprehensive genomic analysis beyond PCR-based methods is necessary to fully characterize editing outcomes.

How might novel gene editing technologies improve CCR5 modification specificity in Macaca fascicularis?

Future improvements in CCR5 editing specificity may come from several technological advances:

• Base editing technologies:

  • Precise C-to-T or A-to-G conversions without double-strand breaks

  • Potentially reduced large-scale chromosomal anomalies

• Prime editing:

  • Programmable "search-and-replace" editing offering precise modifications

  • Reduced off-target effects compared to conventional CRISPR-Cas9

• High-fidelity Cas9 variants:

  • Engineered Cas9 proteins with reduced off-target activity

  • Application of SpCas9-HF1, eSpCas9, or HypaCas9 variants

• Alternative delivery methods:

  • Ribonucleoprotein (RNP) delivery to reduce editing duration and off-target effects

  • Timing optimization to minimize mosaicism

• Improved computational prediction:

  • Enhanced algorithms for off-target prediction

  • Integration of chromatin accessibility data to predict actual off-target likelihood

These advances may address the current limitations of CRISPR-Cas9 editing in macaque embryos, including large-scale deletions, inversions, and mosaicism .

What emerging analytical techniques could enhance detection of CCR5 editing outcomes in Macaca fascicularis?

Emerging analytical techniques that could improve detection of CCR5 editing outcomes include:

• Long-read sequencing:

  • Technologies like PacBio or Oxford Nanopore for improved detection of structural variants

  • Better characterization of large deletions and inversions currently challenging to detect with short-read platforms

• Digital droplet PCR:

  • Improved quantification of editing efficiency

  • Enhanced sensitivity for detecting low-frequency edits

• Single-cell multi-omics:

  • Integrated analysis of genomic and transcriptomic changes

  • Assessment of functional consequences of CCR5 modification at single-cell resolution

• CRISPR-specific sequencing approaches:

  • DISCOVER-Seq for unbiased off-target detection

  • GUIDE-Seq or CIRCLE-Seq for comprehensive off-target profiling

• Advanced bioinformatic pipelines:

  • Integration of multiple structural variant callers

  • Machine learning approaches to distinguish true editing events from sequencing artifacts

These techniques would address the limitations of current methods, where short-read sequencing platforms are "not ideally suited for identifying structural variants" and provide more comprehensive characterization of editing outcomes.