Match!

Directed Evolution of Adenine Base Editors with Increased Activity and Therapeutic Application

Published on Mar 16, 2020in bioRxiv
· DOI :10.1101/2020.03.13.990630
Nicole M. Gaudelli6
Estimated H-index: 6
,
Dieter K. Lam2
Estimated H-index: 2
+ 14 AuthorsAlexander J. Liquori2
Estimated H-index: 2
Abstract
The foundational adenine base editors (e.g. ABE7.10) enable programmable C:G to T:A point mutations but editing efficiencies can be low at challenging loci in primary human cells. Here we further evolve ABE7.10 using a library of adenosine deaminase variants to create ABE8s. At NGG PAM sites, ABE8s result in ~1.5x higher editing at protospacer positions A5-A7 and ~3.2x higher editing at positions A3-A4 and A8-A10 compared with ABE7.10. Non-NGG PAM variants have a ~4.2-fold overall higher on-target editing efficiency than ABE7.10. In human CD34+ cells, ABE8 can recreate a natural allele at the promoter of the gamma-globin genes HBG1 and HBG2, with up to 60% efficiency, causing persistence of fetal hemoglobin. In primary human T cells, ABE8s achieve 98-99% target modification which is maintained when multiplexed across three loci. Delivered as mRNA, ABE8s induce no significant levels of sgRNA-independent off-target adenine deamination in genomic DNA and very low levels of adenine deamination in cellular mRNA.
  • References (49)
  • Citations (2)
References49
Newest
#1Kai Hua (CAS: Chinese Academy of Sciences)H-Index: 5
#2Xiaoping Tao (CAS: Chinese Academy of Sciences)H-Index: 5
Last. Jian-Kang Zhu (CAS: Chinese Academy of Sciences)H-Index: 139
view all 6 authors...
4 CitationsSource
#1Hye-Kyung Lee (NIH: National Institutes of Health)H-Index: 13
#2Harold E. Smith (NIH: National Institutes of Health)H-Index: 12
Last. Lothar Hennighausen (NIH: National Institutes of Health)H-Index: 89
view all 5 authors...
Deaminase base editing has emerged as a tool to install or correct point mutations in the genomes of living cells in a wide range of organisms. However, the genome-wide off-target effects introduced by base editors in the mammalian genome have been examined in only one study. Here, we have investigated the fidelity of cytosine base editor 4 (BE4) and adenine base editors (ABE) in mouse embryos using unbiased whole-genome sequencing of a family-based trio cohort. The same sgRNA was used for BE4 a...
4 CitationsSource
#1S. DepilH-Index: 1
#1Stéphane DepilH-Index: 17
Last. Laurent PoirotH-Index: 10
view all 5 authors...
Autologous chimeric antigen receptor (CAR) T cells have changed the therapeutic landscape in haematological malignancies. Nevertheless, the use of allogeneic CAR T cells from donors has many potential advantages over autologous approaches, such as the immediate availability of cryopreserved batches for patient treatment, possible standardization of the CAR-T cell product, time for multiple cell modifications, redosing or combination of CAR T cells directed against different targets, and decrease...
9 CitationsSource
#1Chun-Qing Song (UMMS: University of Massachusetts Medical School)H-Index: 13
#2Tingting Jiang (UMMS: University of Massachusetts Medical School)H-Index: 2
Last. Wen XueH-Index: 28
view all 15 authors...
In contrast to traditional CRISPR–Cas9 homology-directed repair, base editing can correct point mutations without supplying a DNA-repair template. Here we show in a mouse model of tyrosinaemia that hydrodynamic tail-vein injection of plasmid DNA encoding the adenine base editor (ABE) and a single-guide RNA (sgRNA) can correct an A>G splice-site mutation. ABE treatment partially restored splicing, generated fumarylacetoacetate hydrolase (FAH)-positive hepatocytes in the liver, and rescued weight ...
3 CitationsSource
#1Erica McGrath (CBER: Center for Biologics Evaluation and Research)H-Index: 1
#2Hyunsu Shin (CBER: Center for Biologics Evaluation and Research)H-Index: 1
Last. Zhaohui Ye (CBER: Center for Biologics Evaluation and Research)H-Index: 1
view all 9 authors...
DNA base editors have enabled genome editing without generating DNA double strand breaks. The applications of this technology have been reported in a variety of animal and plant systems, however, their editing specificity in human stem cells has not been studied by unbiased genome-wide analysis. Here we investigate the fidelity of cytidine deaminase-mediated base editing in human induced pluripotent stem cells (iPSCs) by whole genome sequencing after sustained or transient base editor expression...
4 CitationsSource
#1Edward A. Stadtmauer (UPenn: University of Pennsylvania)H-Index: 70
#2Adam D. Cohen (UPenn: University of Pennsylvania)H-Index: 32
Last. Carl H. June (UPenn: University of Pennsylvania)H-Index: 124
view all 31 authors...
2 CitationsSource
#1Julian Grünewald (Harvard University)H-Index: 1
#1Julian Grünewald (Harvard University)H-Index: 2
Last. J. Keith Joung (Harvard University)H-Index: 62
view all 7 authors...
Cytosine or adenine base editors (CBEs or ABEs) can introduce specific DNA C-to-T or A-to-G alterations1–4. However, we recently demonstrated that they can also induce transcriptome-wide guide-RNA-independent editing of RNA bases5, and created selective curbing of unwanted RNA editing (SECURE)-BE3 variants that have reduced unwanted RNA-editing activity5. Here we describe structure-guided engineering of SECURE-ABE variants with reduced off-target RNA-editing activity and comparable on-target DNA...
14 CitationsSource
#1Hye-Kyung Lee (NIH: National Institutes of Health)H-Index: 13
#2Harold E. Smith (NIH: National Institutes of Health)H-Index: 12
Last. Lothar Hennighausen (NIH: National Institutes of Health)H-Index: 89
view all 5 authors...
Deaminase base editing has emerged as a tool to install or correct point mutations in the genomes of living cells in a wide range of organisms and its ultimate success therapeutically depends on its accuracy. Here we have investigated the fidelity of cytosine base editor 4 (BE4) and adenine base editor (ABE) in mouse embryos using unbiased whole genome sequencing of a family-based trio cohort. We demonstrate that BE4-edited mice carry an excess of single-nucleotide variants and deletions compare...
Source
#1Choongil Lee (SNU: Seoul National University)H-Index: 5
#2Dong Hyun Jo (Seoul National University Hospital)H-Index: 13
Last. Sangsu Bae (Hanyang University)H-Index: 15
view all 9 authors...
A nonsense mutation is a substitutive mutation in a DNA sequence that causes a premature termination during translation and produces stalled proteins, resulting in dysfunction of a gene. Although it usually induces severe genetic disorders, there are no definite methods for inducing read through of premature termination codons (PTCs). Here, we present a targeted tool for bypassing PTCs, named CRISPR-pass, that uses CRISPR-mediated adenine base editors. CRISPR-pass, which should be applicable to ...
2 CitationsSource
#1Changyang Zhou (CAS: Chinese Academy of Sciences)H-Index: 5
#2Yidi Sun (CAS: Chinese Academy of Sciences)H-Index: 8
Last. Hui Yang (CAS: Chinese Academy of Sciences)H-Index: 19
view all 14 authors...
Recently developed DNA base editing methods enable the direct generation of desired point mutations in genomic DNA without generating any double-strand breaks1–3, but the issue of off-target edits has limited the application of these methods. Although several previous studies have evaluated off-target mutations in genomic DNA4–8, it is now clear that the deaminases that are integral to commonly used DNA base editors often bind to RNA9–13. For example, the cytosine deaminase APOBEC1—which is used...
16 CitationsSource
Cited By2
Newest
#1Ruth E Hanna (Broad Institute)H-Index: 4
#2Mudra Hegde (Broad Institute)H-Index: 6
Last. Yossef Baidi (Broad Institute)
view all 14 authors...
Understanding the functional consequences of single-nucleotide variants is critical to uncovering the genetic underpinnings of diseases, but technologies to characterize variants are limiting. Here we leverage CRISPR-Cas9 cytosine base editors in pooled screens to scalably assay variants at endogenous loci in mammalian cells. We benchmark the performance of base editors in positive and negative selection screens and identify known loss-of-function mutations in BRCA1 and BRCA2 with high precision...
Source
#1Mitchell G. Kluesner (UMN: University of Minnesota)H-Index: 2
#2Walker S. Lahr (UMN: University of Minnesota)H-Index: 2
Last. Aneesha A. Andrews (UMN: University of Minnesota)H-Index: 1
view all 12 authors...
Base editors allow for precise nucleotide editing without the need for genotoxic double-stranded breaks. Prior work has used base editors to knockout genes by introducing premature stop codons or by disrupting conserved splice-sites, but no direct comparison exists between these methods. Additionally, while base editor mediated disruption of splice sites has been used to shift the functional isoform pool, its utility for gene knockout requires further validation. To address these needs, we devel...
1 CitationsSource