In Silico Analysis of Pathogenic CRB1 Single Nucleotide Variants and Their Amenability to Base Editing as a Potential Lead for Therapeutic Intervention

Mutations in the Crumbs homolog 1 (CRB1) gene cause both autosomal recessive retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA). Since three separate CRB1 isoforms are expressed at meaningful levels in the human retina, base editing shows promise as a therapeutic approach. This retrospective analysis aims to summarise the reported pathogenic CRB1 variants and investigate their amenability to treatment with currently available DNA base editors. Pathogenic single nucleotide variants (SNVs) were extracted from the Leiden open-source variation database (LOVD) and ClinVar database and coded by mutational consequence. They were then analyzed for their amenability to currently available DNA base editors and available PAM sites from a selection of different Cas proteins. Of a total of 1115 unique CRB1 variants, 69% were classified as pathogenic SNVs. Of these, 62% were amenable to currently available DNA BEs. Adenine base editors (ABEs) alone have the potential of targeting 34% of pathogenic SNVs; 19% were amenable to a CBE while GBEs could target an additional 9%. Of the pathogenic SNVs targetable with a DNA BE, 87% had a PAM site for a Cas protein. Of the 33 most frequently reported pathogenic SNVs, 70% were targetable with a base editor. The most common pathogenic variant was c.2843G>A, p.Cys948Arg, which is targetable with an ABE. Since 62% of pathogenic CRB1 SNVs are amenable to correction with a base editor and 87% of these mutations had a suitable PAM site, gene editing represents a promising therapeutic avenue for CRB1-associated retinal degenerations.

Download Full-text

Cytosine base editor 4 but not adenine base editor generates off-target mutations in mouse embryos

Communications Biology ◽

10.1038/s42003-019-0745-3 ◽

2020 ◽

Vol 3 (1) ◽

Cited By ~ 15

Author(s):

Hye Kyung Lee ◽

Harold E. Smith ◽

Chengyu Liu ◽

Michaela Willi ◽

Lothar Hennighausen

Keyword(s):

Point Mutations ◽

Mouse Embryos ◽

Single Nucleotide Variants ◽

Single Nucleotide ◽

Base Editing ◽

Genome Wide ◽

Wide Range ◽

Family Based ◽

Correct Point ◽

Adenine Base

AbstractDeaminase 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 and ABE. We demonstrate that BE4-edited mice carry an excess of single-nucleotide variants and deletions compared to ABE-edited mice and controls. Therefore, an optimization of cytosine base editors is required to improve its fidelity. While the remarkable fidelity of ABE has implications for a wide range of applications, the occurrence of rare aberrant C-to-T conversions at specific target sites needs to be addressed.

Download Full-text

Cytosine, but not adenine, base editors induce genome-wide off-target mutations in rice

Science ◽

10.1126/science.aaw7166 ◽

2019 ◽

pp. eaaw7166 ◽

Cited By ~ 77

Author(s):

Shuai Jin ◽

Yuan Zong ◽

Qiang Gao ◽

Zixu Zhu ◽

Yanpeng Wang ◽

...

Keyword(s):

High Fidelity ◽

Single Nucleotide Variants ◽

Disease Treatment ◽

Single Nucleotide ◽

Genetic Changes ◽

Base Editing ◽

Guide Rna ◽

Genome Wide ◽

Adenine Base

Cytosine and adenine base editors (CBEs and ABEs) are promising new tools for achieving the precise genetic changes required for disease treatment and trait improvement. However, genome-wide and unbiased analyses of their off-target effects in vivo are still lacking. Our whole genome sequencing (WGS) analysis of rice plants treated with BE3, high-fidelity BE3 (HF1-BE3), or ABE revealed that BE3 and HF1-BE3, but not ABE, induce substantial genome-wide off-target mutations, which are mostly the C→T type of single nucleotide variants (SNVs) and appear to be enriched in genic regions. Notably, treatment of rice with BE3 or HF1-BE3 in the absence of single-guide RNA also results in the rise of genome-wide SNVs. Thus, the base editing unit of BE3 or HF1-BE3 needs to be optimized in order to attain high fidelity.

Download Full-text

Computational detection, analysis and interpretations of genomic variants in human diseases associated GENEMDM 2

Biomedical Letters ◽

10.47262/bl/7.2.20210705 ◽

2021 ◽

Vol 7 (2) ◽

pp. 141-154

Keyword(s):

Computational Methods ◽

High Throughput Sequencing ◽

Cancer Susceptibility ◽

In Silico Analysis ◽

Human Diseases ◽

Nucleotide Polymorphisms ◽

Single Nucleotide Variants ◽

Mdm2 Gene ◽

Single Nucleotide ◽

Computational Tools

Most of the mutations described in human MDM2 are tolerated without significantly disrupting the corresponding structural or molecular function. However, some of them are associated with a variety of human diseases, including cancer. Numerous computational methods have been developed to predict the effects of missense single nucleotide variants (SNVs). The non-synonymous single nucleotide polymorphisms affect the function of XRCC1, which impairs the ability to repair DNA and therefore increases the risk of diseases such as cancer. In this study, sequence and structure-based computational tools were used to screen the total listed coding SNPs of the MDM2 gene in order to recognize and describe them. The potential 6 ns SNP of MDM2 were identified from 29 ns SNP by consistent analysis using computational tools PolyPhen 2, SIFT, PANTHER and cSNP. The computational methods were used to systematically classify functional mutations in the regulatory and coding regions that modify the expression and function of the MDM2 enzyme. The HOPE project also made it possible to elaborate the structural effects of the substitutions of amino acids. In silico analysis predicted that rs759244097 is harmful. This study concluded that identifying this SNP will help to determine an individual's cancer susceptibility, prognosis and further treatment. Furthermore, current high-throughput sequencing efforts and the need for extensive interpretation of protein sequence variants requires more efficient and accurate computational methods in the coming years.

Download Full-text

Unexpected CRISPR off-target mutation pattern in vivo are not typically germline-like

10.1101/193565 ◽

2017 ◽

Cited By ~ 1

Author(s):

Zhiting Wei ◽

Funan He ◽

Guohui Chuai ◽

Hanhui Ma ◽

Zhixi Su ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Whole Genome ◽

Single Nucleotide Variants ◽

Single Nucleotide ◽

Cas9 Nuclease ◽

Mutation Pattern ◽

And Control ◽

Selection Of

To the EditorSchaefer et al.1 (referred to as Study_1) recently presented the provocative conclusion that CRISPR-Cas9 nuclease can induce many unexpected off-target mutations across the genome that arise from the sites with poor homology to the gRNA. As Wilson et al.2 pointed out, however, the selection of a co-housed mouse as the control is insufficient to attribute the observed mutation differences between the CRISPR-treated mice and control mice. Therefore, the causes of these mutations need to be further investigated. In 2015, Iyer et al.3 (referred to as Study_2) used Cas9 and a pair of sgRNAs to mutate the Ar gene in vivo and off-target mutations were investigated by comparison the control mice and the offspring of the modified mice. After analyzing the whole genome sequencing (WGS) of the offspring and the control mice, they claimed that off-target mutations are rare from CRISPR-Cas9 engineering. Notably, their study only focused on indel off-target mutations. We re-analyzed the WGS data of these two studies and detected both single nucleotide variants (SNVs) and indel mutations.

Download Full-text

Cytosine but not adenine base editor generates mutations in mice

10.1101/731927 ◽

2019 ◽

Cited By ~ 1

Author(s):

Hye Kyung Lee ◽

Harold E. Smith ◽

Chengyu Liu ◽

Michaela Willi ◽

Lothar Hennighausen

Keyword(s):

Point Mutations ◽

Living Cells ◽

Mouse Embryos ◽

Single Nucleotide Variants ◽

Single Nucleotide ◽

Base Editing ◽

Wide Range ◽

Family Based ◽

Correct Point ◽

Adenine Base

ABSTRACTDeaminase 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 compared to ABE-edited mice and controls.

Download Full-text

In silico analysis of missense Single Nucleotide Variants (SNVs) in HBB gene associated with the β-thalassemia

Gene Reports ◽

10.1016/j.genrep.2021.101019 ◽

2021 ◽

Vol 23 ◽

pp. 101019

Author(s):

Malik Siddique Mahmood ◽

Saba Irshad ◽

Umme Kalsoom ◽

Hina Batool ◽

Sana Batool ◽

...

Keyword(s):

In Silico ◽

In Silico Analysis ◽

Single Nucleotide Variants ◽

Single Nucleotide ◽

Silico Analysis

Download Full-text

Insertion of the protective APP A673T mutation by CRISPR/Cas9 base editing or PRIME editing

Innovation in Aging ◽

10.1093/geroni/igab046.2505 ◽

2021 ◽

Vol 5 (Supplement_1) ◽

pp. 669-669

Author(s):

Jacques P Tremblay ◽

Antoine Guyon ◽

Joël Rousseau ◽

Guillaume Tremblay ◽

Francis-Gabriel Begin ◽

...

Keyword(s):

Beta Amyloid ◽

Sporadic Alzheimer’S Disease ◽

Single Nucleotide ◽

Amyloid Peptides ◽

Base Editing ◽

Beta Secretase ◽

Human Neurons ◽

Nucleotide Mutation ◽

App Gene ◽

Therapeutic Avenue

Abstract There is currently no treatment for Alzheimer disease (AD). However, the Icelandic mutation in the APP gene (A673T) has been shown to confer a protection against the onset and development of AD (Jonsson et al. Nature 2012). This single nucleotide mutation in APP exon 16 reduces the cleavage of the APP protein by the beta-secretase by 40% thus preventing the development of AD even in persons more than 95 years old. Our research group has initially shown that the presence of the A673T mutation in an APP gene reduced the secretion of beta-amyloid peptides even if there is also a FAD mutation in the gene. This is the case for 14 different FAD mutations. We have used CRISPR/Cas9 base editing and PRIME editing technologies to insert the A673T mutation in the APP gene. We have compared several different cytidine base editor complexes to achieve the most effective and accurate genome modification possible in HEK293T cells and in SH-SY5Y neuroblastomas. The insertion of the A673T mutation in cells containing the London mutation reduced the secretion of beta-amyloid peptides. We are currently using lentiviral vectors to infect neurons from a mouse model and human neurons induced from fibroblasts of a patient with the London mutation. The insertion of the protective Icelandic mutation in the APP gene using these editing technologies opens a new potential therapeutic avenue not only for Familial Alzheimer’s diseases but also for sporadic Alzheimer’s disease.

Download Full-text

Complementary information on single nucleotide variants, INDELs and functional translocations can be obtained with RNAseq using different library preparations

10.1101/301010 ◽

2018 ◽

Author(s):

Riccardo Panero ◽

Maddalena Arigoni ◽

Martina Olivero ◽

Francesca Cordero ◽

Alessandro Weisz ◽

...

Keyword(s):

The Other ◽

Rna Seq ◽

Single Nucleotide Variants ◽

Complementary Information ◽

Single Nucleotide ◽

Sequencing Platform ◽

Variant Frequency ◽

Selection Protocol ◽

Variant Detection ◽

Selection Of

AbstractBackgroundRNA-seq represents an attractive methodology for the detection of functional genomic variants because it allows the integration of variant frequency and their expression. However, although specific statistic frameworks have been designed to detect SNVs/INDELS/gene fusions in RNA-seq data, very little has been done to understand the effect of library preparation protocols on transcript variant detection in RNA-seq data.ResultsHere, we compared RNA-seq results obtained on short reads sequencing platform with two protocols: one based on polyA+ RNA selection protocol (POLYA) and the other based on exonic regions capturing protocol (ACCESS). Our data indicate that ACCESS detects 10% more coding SNV/INDELs with respect to POLYA, making this protocol more suitable for this goal. Furthermore, ACCESS requires less reads for coding SNV detection with respect to POLYA. On the other hand, if the analysis aims at identifying SNV/INDELs also in the 5’and 3’ UTRs, POLYA is definitively the preferred method. No particular advantage comes from the usage of ACCESS or POLYA in the detection of fusion transcripts.ConclusionData show that a careful selection of the “wet” protocol adds specific features that cannot be obtained with bioinformatics alone.

Download Full-text

Base editing generates substantial off-target single nucleotide variants

10.1101/480145 ◽

2018 ◽

Cited By ~ 5

Author(s):

Erwei Zuo ◽

Yidi Sun ◽

Wu Wei ◽

Tanglong Yuan ◽

Wenqin Ying ◽

...

Keyword(s):

Mammalian Cells ◽

Biomedical Application ◽

Spontaneous Mutation ◽

Great Promise ◽

Single Nucleotide Variants ◽

Spontaneous Mutation Rate ◽

Single Nucleotide ◽

Base Editing ◽

Genome Wide ◽

Target Effects

AbstractGenome editing tools including CRISPR/Cas9 and base editors hold great promise for correcting pathogenic mutations. Unbiased genome-wide off-target effects of the editing in mammalian cells is required before clinical applications, but determination of the extent of off-target effects has been difficult due to the existence of single nucleotide polymorphisms (SNPs) in individuals. Here, we developed a method named GOTI (Genome-wide Off-target analysis by Two-cell embryo Injection) to detect off-target mutations without interference of SNPs. We applied GOTI to both the CRISPR-Cas9 and base editing (BE3) systems by editing one blastomere of the two-cell mouse embryo and then compared whole genome sequences of progeny-cell populations at E14.5 stage. Sequence analysis of edited and non-edited cell progenies showed that undesired off-target single nucleotide variants (SNVs) are rare (average 10.5) in CRISPR-edited mouse embryos, with a frequency close to the spontaneous mutation rate. By contrast, BE3 editing induced over 20-fold higher SNVs (average 283), raising the concern of using base-editing approaches for biomedical application.

Download Full-text

Genome scale analysis of pathogenic variants targetable for single base editing

BMC Medical Genomics ◽

10.1186/s12920-020-00735-8 ◽

2020 ◽

Vol 13 (S8) ◽

Author(s):

Alexander V. Lavrov ◽

Georgi G. Varenikov ◽

Mikhail Yu Skoblov

Keyword(s):

Protein Engineering ◽

Targeted Therapies ◽

Human Diseases ◽

Scale Analysis ◽

Single Nucleotide Variants ◽

Single Nucleotide ◽

Single Base ◽

Base Editing ◽

Pathogenic Variants ◽

Genome Scale

Abstract Background Single nucleotide variants account for approximately 90% of all known pathogenic variants responsible for human diseases. Recently discovered CRISPR/Cas9 base editors can correct individual nucleotides without cutting DNA and inducing double-stranded breaks. We aimed to find all possible pathogenic variants which can be efficiently targeted by any of the currently described base editors and to present them for further selection and development of targeted therapies. Methods ClinVar database (GRCh37_clinvar_20171203) was used to search and select mutations available for current single-base editing systems. We included only pathogenic and likely pathogenic variants for further analysis. For every potentially editable mutation we checked the presence of PAM. If a PAM was found, we analyzed the sequence to find possibility to edit only one nucleotide without changing neighboring nucleotides. The code of the script to search Clinvar database and to analyze the sequences was written in R and is available in the appendix. Results We analyzed 21 editing system currently reported in 9 publications. Every system has different working characteristics such as the editing window and PAM sequence. C > T base editors can precisely target 3196 mutations (46% of all pathogenic T > C variants), and A > G editors – 6900 mutations (34% of all pathogenic G > A variants). Conclusions Protein engineering helps to develop new enzymes with a narrower window of base editors as well as using new Cas9 enzymes with different PAM sequences. But, even now the list of mutations which can be targeted with currently available systems is huge enough to choose and develop new targeted therapies.

Download Full-text