B - Gene Targeting and Gene Correction -> B2 - Gene Targeting and Gene Correction – In Vitro Studies (Basic development of novel technologies for genome editing, with or without site-specific endonuclease.
97: CHANGE-seq-BE Enables Sensitive and Unbiased Genome-Wide Profiling of Adenine Base Editors In Vitro
Type: Oral Abstract Session
Presentation Details
Session Title: Genome Editing Therapies & Safety I
Location: Room 515 AB
Start Time: 5/17/2023 16:15
End Time: 5/17/2023 16:30
Base editors can correct pathogenic single-nucleotide variants without introducing DNA double-stranded breaks and have tremendous potential to become a new class of safe and effective genomic medicines. However, current methods for understanding the safety and genome-wide activity of base editors have limitations. Some have limited sensitivity as they do not enrich for base editor modified genomic DNA and others are experimentally biased by computational pre-selection of candidate off-target sites.
To develop a method for direct, sensitive, unbiased, genome-wide discovery of base editor off-target sites we adapted CHANGE-seq, a scalable biochemical method for defining Cas9 nuclease genome-wide activity, to base editors (CHANGE-seq-BE). We used Tn5 tagmentation and exonuclease selection to generate libraries of highly purified genomic DNA circles. CHANGE-seq-BE enables the selective sequencing of adenine base editor modified genomic DNA by enzymatically processing circularized genomic DNA that is nicked and deaminated by base editors into full DNA DSBs for adapter ligation and sequencing. To systematically evaluate the genome-wide off-target activity of ABE8e in vitro, we performed CHANGE-seq-BE with sgRNAs targeted to seven therapeutic relevant target sites (PCSK9, HBG1/HBG2 -198, CD7, CIITA, PDCD1, B2M and CBLB). We found CHANGE-seq-BE read counts were strongly correlated between independent CHANGE-seq-BE technical replicates (R2>0.9). To validate the sensitivity of CHANGE-seq-BE for identifying sites of bona fide cellular off-target base editing, we selected 105 off-target sites from five sgRNA target sites, for analysis by multiplex targeted sequencing in ABE8e base edited cells. Of the 105 CHANGE-seq-BE detected sites we examined, we confirmed 26 (24.7%) by targeted sequencing, with activity ranging from 0.67-88.8%, demonstrating that our method can sensitively detect ABE off-target activity. Next, to determine whether CHANGE-seq-BE could identify base editor-specific off-target activity not detected by nuclease-specific methods, we compared sites identified by CHANGE-seq-BE and by CIRCLE-seq. For a sgRNA targeted to the HBB gene, we found that CHANGE-seq-BE identified all known off-target sites as well as an additional ~53% previously unknown bona fide off-targets than our initial screen with CIRCLE-seq (Newby et. al, 2021). Furthermore, percentile ranks of validated off-target sites were higher in CHANGE-seq-BE compared to CIRCLE-seq. Taken together, our results demonstrate that direct base-editor specific off-target discovery methods outperform indirect nuclease off-target discovery to comprehensively identify ABE genome-wide off-target activity. In summary, CHANGE-seq-BE enables the rapid characterization of ABE genome-wide activity at a scale not previously achievable by other methods and represents a simple, rapid, sensitive and unbiased method to routinely define genome-wide base editing activity for research and therapeutic applications.
Cicera R. Lazzarotto1,2, Yichao Li1, Varun Katta1, Elizabeth Urbina Obregon1, Jonathan Yen1, Mitchell J. Weiss1, Shengdar Q. Tsai1
1St. Jude Children's Research Hospital, Memphis, TN,2Beam Therapeutics, Cambridge, MA
C.R. Lazzarotto: 1; Commercial Interest i.e. Company X; Beam Therapeutics. 1; What was received? i.e. Honorarium; Salary. 1; For what role? i.e. Speaker; Senior Scientist.
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