Continuous directed evolution methods allow the key steps of evolution-gene diversification, selection, and replication-to proceed in the laboratory with minimal researcher intervention. As a result, ...continuous evolution can find solutions much more quickly than traditional discrete evolution methods. Continuous evolution also enables the exploration of longer and more numerous evolutionary trajectories, increasing the likelihood of accessing solutions that require many steps through sequence space and greatly facilitating the iterative refinement of selection conditions and targeted mutagenesis strategies. Here we review the historical advances that have expanded continuous evolution from its earliest days as an experimental curiosity to its present state as a powerful and surprisingly general strategy for generating tailor-made biomolecules, and discuss more recent improvements with an eye to the future.
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GEOZS, IJS, IMTLJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK, ZAGLJ
The targeted deletion, replacement, integration or inversion of genomic sequences could be used to study or treat human genetic diseases, but existing methods typically require double-strand DNA ...breaks (DSBs) that lead to undesired consequences, including uncontrolled indel mixtures and chromosomal abnormalities. Here we describe twin prime editing (twinPE), a DSB-independent method that uses a prime editor protein and two prime editing guide RNAs (pegRNAs) for the programmable replacement or excision of DNA sequences at endogenous human genomic sites. The two pegRNAs template the synthesis of complementary DNA flaps on opposing strands of genomic DNA, which replace the endogenous DNA sequence between the prime-editor-induced nick sites. When combined with a site-specific serine recombinase, twinPE enabled targeted integration of gene-sized DNA plasmids (>5,000 bp) and targeted sequence inversions of 40 kb in human cells. TwinPE expands the capabilities of precision gene editing and might synergize with other tools for the correction or complementation of large or complex human pathogenic alleles.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Epitope-specific enzymes are powerful tools for site-specific protein modification but generally require genetic manipulation of the target protein. Here, we describe the laboratory evolution of the ...bacterial transpeptidase sortase A to recognize the LMVGG sequence in endogenous amyloid-β (Aβ) protein. Using a yeast display selection for covalent bond formation, we evolved a sortase variant that prefers LMVGG substrates from a starting enzyme that prefers LPESG substrates, resulting in a >1,400-fold change in substrate preference. We used this evolved sortase to label endogenous Aβ in human cerebrospinal fluid, enabling the detection of Aβ with sensitivities rivaling those of commercial assays. The evolved sortase can conjugate a hydrophilic peptide to Aβ
, greatly impeding the ability of the resulting protein to aggregate into higher-order structures. These results demonstrate laboratory evolution of epitope-specific enzymes toward endogenous targets as a strategy for site-specific protein modification without target gene manipulation and enable potential future applications of sortase-mediated labeling of Aβ peptides.
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GEOZS, IJS, IMTLJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK, ZAGLJ
CPAF (chlamydial protease-like activity factor) is a Chlamydia trachomatis protease that is translocated into the host cytosol during infection. CPAF activity results in dampened host inflammation ...signaling, cytoskeletal remodeling, and suppressed neutrophil activation. Although CPAF is an emerging antivirulence target, its catalytic mechanism has been unexplored to date. Steady state kinetic parameters were obtained for recombinant CPAF with vimentin-derived peptide substrates using a high-performance liquid chromatography-based discontinuous assay (k cat = 45 ± 0.6 s–1; k cat/K m = 0.37 ± 0.02 μM–1 s–1) or a new fluorescence-based continuous assay (k cat = 23 ± 0.7 s–1; k cat/K m = 0.29 ± 0.03 μM–1 s–1). Residues H105, S499, E558, and newly identified D103 were found to be indispensable for autoproteolytic processing by mutagenesis, while participation of C500 was ruled out despite its proximity to the S499 nucleophile. Pre-steady state kinetics indicated a burst kinetic profile, with fast acylation (k acyl = 110 ± 2 s–1) followed by slower, partially rate-limiting deacylation (k deacyl = 57 ± 1 s–1). Both k cat– and k cat/K m–pH profiles showed single acidic limb ionizations with pK a values of 6.2 ± 0.1 and 6.5 ± 0.1, respectively. A forward solvent deuterium kinetic isotope effect of 2.6 ± 0.1 was observed for D2O k cat app, but a unity effect was found for D2O k cat/K m app. The k cat proton inventory was linear, indicating transfer of a single proton in the rate-determining transition state, most likely from H105. Collectively, these data provide support for the classification of CPAF as a serine protease and provide a mechanistic foundation for the future design of inhibitors.
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IJS, KILJ, NUK, PNG, UL, UM
Sickle cell disease (SCD) is caused by a mutation in the β-globin gene HBB
. We used a custom adenine base editor (ABE8e-NRCH)
to convert the SCD allele (HBB
) into Makassar β-globin (HBB
), a ...non-pathogenic variant
. Ex vivo delivery of mRNA encoding the base editor with a targeting guide RNA into haematopoietic stem and progenitor cells (HSPCs) from patients with SCD resulted in 80% conversion of HBB
to HBB
. Sixteen weeks after transplantation of edited human HSPCs into immunodeficient mice, the frequency of HBB
was 68% and hypoxia-induced sickling of bone marrow reticulocytes had decreased fivefold, indicating durable gene editing. To assess the physiological effects of HBB
base editing, we delivered ABE8e-NRCH and guide RNA into HSPCs from a humanized SCD mouse
and then transplanted these cells into irradiated mice. After sixteen weeks, Makassar β-globin represented 79% of β-globin protein in blood, and hypoxia-induced sickling was reduced threefold. Mice that received base-edited HSPCs showed near-normal haematological parameters and reduced splenic pathology compared to mice that received unedited cells. Secondary transplantation of edited bone marrow confirmed that the gene editing was durable in long-term haematopoietic stem cells and showed that HBB
-to-HBB
editing of 20% or more is sufficient for phenotypic rescue. Base editing of human HSPCs avoided the p53 activation and larger deletions that have been observed following Cas9 nuclease treatment. These findings point towards a one-time autologous treatment for SCD that eliminates pathogenic HBB
, generates benign HBB
, and minimizes the undesired consequences of double-strand DNA breaks.
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GEOZS, IJS, IMTLJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK, ZAGLJ
While CAR-T cell therapy represents a major advance in personalized immunotherapy, the autologous nature of commercially available CAR-T products have delayed its broad application beyond a subset of ...hematological malignancies. In particular, manufacturing autologous CAR-T therapies is costly and time-consuming, and success relies on the fitness of a patient's cells. An allogeneic off-the-shelf CAR-T product could overcome these cell quality and quantity issues and could be accessed on-demand. However, a successful allogeneic CAR-T product will require multiplex gene knockout in addition to stable CAR integration to prevent graft-versus-host disease (GvHD) and graft rejection by the patient's immune system. Strategies for the delivery and expression of CAR transgenes include semi-random integration using lentivirus or transposons, which risk unintended gene disruption or activation. Targeted integration can be achieved using nucleases in combination with a template for homology-directed repair (HDR), but efficiency of this approach is generally low and there are risks associated with the induction of double-strand breaks (DSBs), including p53 activation and chromothripsis. This is further complicated in a situation where multiplex editing is required, as multiple DSBs can lead to gross chromosomal rearrangements. Alternatively, base editors have been used for targeted gene disruption without the induction of DSBs via single nucleotide conversion to disrupt a splice site or introduce a single premature stop (pmSTOP) codon. However, this approach is limited to transition mutations and cannot be used to integrate large genetic cargo, which is required to generate CAR-T cells. Prime Editing (PE) can be used for precise and programmable gene disruption via multiple strategies, including introduction of multiple pmSTOP codons, splice site disruption, frameshift mutations, or deletion of large segments of regulatory or coding sequence. Further, Prime-Assisted Site-Specific Integrase Gene Editing (PASSIGE) can be used to precisely and flexibly integrate large genetic cargo at a specific locus. Together, PE-mediated gene knock out and PASSIGE can be tailored to generate a more broadly applicable, potentially safer, and more effective CAR-T cell product. To evaluate the efficacy of an all-PE non-viral approach to allogeneic CAR-T cell generation, PASSIGE and PE mediated gene knockout were used to generate CD19-CAR-T cells. Multiplex PE precisely disrupted expression of the endogenous T cell receptor alpha constant ( TRAC) and beta-2-microglobulin ( B2M) loci in over 90% of human T cells. Co-delivery of a non-viral DNA donor template with PASSIGE editing components resulted in targeted integration of a 3.5 kb CD19-CAR transgene expression cassette at the TRAC locus in over 60% of the T cells, with no observed impact on T cell viability, phenotype, or functionality. CD19 CAR-T cells generated using PASSIGE show potent anti-tumor activity and cytokine production in response to CD19 + tumor cell lines in vitro and in vivo. The PASSIGE-generated CD19 CAR-T cells reduce tumor burden and prolong survival of mice bearing CD19 + tumors. These results show that a PE platform can be used to generate multiplex edited CAR-T cells without the need for viral vectors and without causing DSBs. This modular, one-step, non-viral delivery Prime Editing platform expands the applicability of T cell therapies for the treatment of tumors and immune diseases.
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IJS, KILJ, KISLJ, NUK, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ
Sickle cell disease (SCD) is caused by a mutation in the β-globin gene
HBB
1
. We used a custom adenine base editor (ABE8e-NRCH)
2
,
3
to convert the SCD allele (
HBB
S
) to Makassar β-globin (
HBB
G
...), a non-pathogenic variant
4
,
5
.
Ex vivo
delivery of mRNA encoding base editor with a targeting guide RNA into hematopoietic stem and progenitor cells (HSPCs) from SCD patients resulted in 80%
HBB
S
-to-
HBB
G
conversion. Sixteen weeks after transplantation of edited human HSPCs into immunodeficient mice,
HBB
G
frequency was 68% and bone marrow reticulocytes demonstrated a 5-fold decrease in hypoxia-induced sickling, indicating durable editing. To assess the physiological effects of
HBB
S
base editing, we delivered ABE8e-NRCH and guide RNA into HSPCs from a humanized SCD mouse
6
, followed by transplantation into irradiated mice. After sixteen weeks, Makassar β-globin represented 79% of β-globin protein in blood and hypoxia-induced sickling was reduced 3-fold. Mice receiving base-edited HSPCs showed rescue of hematologic parameters to near-normal levels and reduced splenic pathology compared to unedited controls. Secondary transplantation of edited bone marrow confirmed durable editing of long-term hematopoietic stem cells and revealed that ≥20%
HBB
S
-to-
HBB
G
editing is sufficient for phenotypic rescue. Base editing of human HSPCs avoided p53 activation and larger deletions observed following Cas9 nuclease treatment. These findings suggest a one-time autologous treatment for SCD that eliminates pathogenic
HBB
S
, generates benign
HBB
G
, and minimizes undesired consequences of double-strand DNA breaks.
Full text
Available for:
GEOZS, IJS, IMTLJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK, ZAGLJ
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