Enhancing genome editing in hPSCs through dual inhibition of DNA damage response and repair pathways.

Precise genome editing is crucial for establishing isogenic human disease models and ex vivo stem cell therapy from the patient-derived hPSCs. Unlike Cas9-mediated knock-in, cytosine base editor and prime editor achieve the desirable gene correction without inducing DNA double strand breaks. However, hPSCs possess highly active DNA repair pathways and are particularly susceptible to p53-dependent cell death. These unique characteristics impede the efficiency of gene editing in hPSCs. Here, we demonstrate that dual inhibition of p53-mediated cell death and distinct activation of the DNA damage repair system upon DNA damage by cytosine base editor or prime editor additively enhanced editing efficiency in hPSCs. The BE4stem system comprised of p53DD, a dominant negative p53, and three UNG inhibitor, engineered to specifically diminish base excision repair, improves cytosine base editor efficiency in hPSCs. Addition of dominant negative MLH1 to inhibit mismatch repair activity and p53DD in the conventional prime editor system also significantly enhances prime editor efficiency in hPSCs. Thus, combined inhibition of the distinct cellular cascades engaged in hPSCs upon gene editing could significantly enhance precise genome editing in these cells.

Direct measurement of engineered cancer mutations and their transcriptional phenotypes in single cells.

Genome sequencing studies have identified numerous cancer mutations across a wide spectrum of tumor types, but determining the phenotypic consequence of these mutations remains a challenge. Here, we developed a high-throughput, multiplexed single-cell technology called TISCC-seq to engineer predesignated mutations in cells using CRISPR base editors, directly delineate their genotype among individual cells and determine each mutation's transcriptional phenotype. Long-read sequencing of the target gene's transcript identifies the engineered mutations, and the transcriptome profile from the same set of cells is simultaneously analyzed by short-read sequencing. Through integration, we determine the mutations' genotype and expression phenotype at single-cell resolution. Using cell lines, we engineer and evaluate the impact of >100 TP53 mutations on gene expression. Based on the single-cell gene expression, we classify the mutations as having a functionally significant phenotype.

METTL3 regulates alternative splicing of cell cycle-related genes via crosstalk between mRNA m6A modifications and splicing factors.

N6-methyladenosine (m6A) modification in RNA affects various aspects of RNA metabolism and regulates gene expression. This modification is modulated by many regulatory proteins, such as m6A methyltransferases (writers), m6A demethylases (erasers), and m6A-binding proteins (readers). Previous studies have suggested that alterations in m6A regulatory proteins induce genome-wide alternative splicing in many cancer cells. However, the functional effects and molecular mechanisms of m6A-mediated alternative splicing have not been fully elucidated. To understand the consequences of this modification on RNA splicing in cancer cells, we performed RNA sequencing and analyzed alternative splicing patterns in METTL3-knockdown osteosarcoma U2OS cells. We detected 1,803 alternatively spliced genes in METTL3-knockdown cells compared to the controls and found that cell cycle-related genes were enriched in differentially spliced genes. A comparison of the published MeRIP-seq data for METTL14 with our RNA sequencing data revealed that 70-87% of alternatively spliced genes had an m6A peak near 1 kb of alternative splicing sites. Among the 19 RNA-binding proteins enriched in alternative splicing sites, as revealed by motif analysis, expression of SFPQ highly correlated with METTL3 expression in 12,839 TCGA pan-cancer patients. We also found that cell cycle-related genes were enriched in alternatively spliced genes of other cell lines with METTL3 knockdown. Taken together, we suggest that METTL3 regulates m6A-dependent alternative splicing, especially in cell cycle-related genes, by regulating the functions of splicing factors such as SFPQ.

High-Fidelity Cytosine Base Editing in a GC-Rich Corynebacterium glutamicum with Reduced DNA Off-Target Editing Effects.

Genome editing technology is a powerful tool for programming microbial cell factories. However, rat APOBEC1-derived cytosine base editor (CBE) that converts C•G to T•A at target genes induced DNA off-targets, regardless of single-guide RNA (sgRNA) sequences. Although the high efficiencies of the bacterial CBEs have been developed, a risk of unidentified off-targets impeded genome editing for microbial cell factories. To address the issues, we demonstrate the genome engineering of Corynebacterium glutamicum as a GC-rich model industrial bacterium by generating premature termination codons (PTCs) in desired genes using high-fidelity cytosine base editors (CBEs). Through this CBE-STOP approach of introducing specific cytosine conversions, we constructed several single-gene-inactivated strains for three genes (ldh, idsA, and pyc) with high base editing efficiencies of average 95.6% (n = 45, C6 position) and the highest success rate of up to 100% for PTCs and ultimately developed a strain with five genes (ldh, actA, ackA, pqo, and pta) that were inactivated sequentially for enhancing succinate production. Although these mutant strains showed the desired phenotypes, whole-genome sequencing (WGS) data revealed that genome-wide point mutations occurred in each strain and further accumulated according to the duration of CBE plasmids. To lower the undesirable mutations, high-fidelity CBEs (pCoryne-YE1-BE3 and pCoryne-BE3-R132E) was employed for single or multiplexed genome editing in C. glutamicum, resulting in drastically reduced sgRNA-independent off-targets. Thus, we provide a CRISPR-assisted bacterial genome engineering tool with an average high efficiency of 90.5% (n = 76, C5 or C6 position) at the desired targets. IMPORTANCE Rat APOBEC1-derived cytosine base editor (CBE) that converts C•G to T•A at target genes induced DNA off-targets, regardless of single-guide RNA (sgRNA) sequences. Although the high efficiencies of bacterial CBEs have been developed, a risk of unidentified off-targets impeded genome editing for microbial cell factories. To address the issues, we identified the DNA off-targets for single and multiple genome engineering of the industrial bacterium Corynebacterium glutamicum using whole-genome sequencing. Further, we developed the high-fidelity (HF)-CBE with significantly reduced off-targets with comparable efficiency and precision. We believe that our DNA off-target analysis and the HF-CBE can promote CRISPR-assisted genome engineering over conventional gene manipulation tools by providing a markerless genetic tool without need for a foreign DNA donor.

Adenine base editor engineering reduces editing of bystander cytosines.

Adenine base editors (ABEs) catalyze specific A-to-G conversions at genomic sites of interest. However, ABEs also induce cytosine deamination at the target site. To reduce the cytosine editing activity, we engineered a commonly used adenosine deaminase, TadA7.10, and found that ABE7.10 with a D108Q mutation in TadA7.10 exhibited tenfold reduced cytosine deamination activity. The D108Q mutation also reduces cytosine deamination activity in two recently developed high-activity versions of ABE, ABE8e and ABE8s, and is compatible with V106W, a mutation that reduces off-target RNA editing. ABE7.10 containing a P48R mutation displayed increased cytosine deamination activity and a substantially reduced adenine editing rate, yielding a TC-specific base editing tool for TC-to-TT or TC-to-TG conversions that broadens the utility of base editors.