CHANGE-seq reveals genetic and epigenetic effects on CRISPR–Cas9 genome-wide activity

Lazzarotto, Cícera R.; Malinin, Nikolay L.; Li, Yichao; Zhang, Ruochi; Yang, Yang; Lee, GaHyun; Cowley, Eleanor; He, Yanghua; X, Lan; Jividen, Kasey; Katta, Varun; Kolmakova, Natalia G.; Petersen, Christopher T.; Qi, Qian; Strelcov, Evgheni; Maragh, Samantha; Krenciute, Giedre; Ma, Jian; Cheng, Yong; Tsai, Shengdar Q.

doi:10.1038/s41587-020-0555-7

Cited by 171 publications

(256 citation statements)

References 51 publications

(55 reference statements)

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“…4G, H thus reports the number of potential off-target sites for each mismatch number for the human and mouse gRNAs. Future implementations of CRISPR mediated B cell engineering in the clinical setting may further require a deep unbiased analysis of off-target cleavage distribution 19 . The ultimate cleavage site in the human IgH J-C intron can then be chosen (Supplementary Data 1) based on it being associated with a minimal off target cleavage profile.…”

Section: Resultsmentioning

confidence: 99%

Engineered B cells expressing an anti-HIV antibody enable memory retention, isotype switching and clonal expansion

et al. 2020

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HIV viremia can be controlled by chronic antiretroviral therapy. As a potentially single-shot alternative, B cells engineered by CRISPR/Cas9 to express anti-HIV broadly neutralizing antibodies (bNAbs) are capable of secreting high antibody titers. Here, we show that, upon immunization of mice, adoptively transferred engineered B cells home to germinal centers (GC) where they predominate over the endogenous response and differentiate into memory and plasma cells while undergoing class switch recombination (CSR). Immunization with a high affinity antigen increases accumulation in GCs and CSR rates. Boost immunization increases the rate of engineered B cells in GCs and antibody secretion, indicating memory retention. Finally, antibody sequences of engineered B cells in the spleen show patterns of clonal selection. Therefore, B cells can be engineered into what could be a living and evolving drug.

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Section: Resultsmentioning

confidence: 99%

Engineered B cells expressing an anti-HIV antibody enable memory retention, isotype switching and clonal expansion

et al. 2020

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“…Our comprehensive benchmark study of these existing resources provides insightful guidance for off-target effect research in four aspects: (i) The benchmarking of experimental CRISPR off-target detection techniques indicated that the gRNA specificity verifies in different experimental OTS detection techniques, resulting from their different experiment categories, DSBs detecting sensitivities, and even the developing times. A recent study provided a new in vitro genome-wide OTS technique called CHANGE-seq ( 42 ), which was reported to perform better than CIRCLE-seq in sequencing efficacy and parallel experiments. (ii) CRISPR cleavage specificity is heterogeneous in different cell types, resulting from their different genetic and epigenetic information.…”

Section: Discussionmentioning

confidence: 99%

Benchmarking and integrating genome-wide CRISPR off-target detection and prediction

Yan

Xue

Chuai

et al. 2020

Nucleic Acids Research

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Systematic evaluation of genome-wide Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) off-target profiles is a fundamental step for the successful application of the CRISPR system to clinical therapies. Many experimental techniques and in silico tools have been proposed for detecting and predicting genome-wide CRISPR off-target profiles. These techniques and tools, however, have not been systematically benchmarked. A comprehensive benchmark study and an integrated strategy that takes advantage of the currently available tools to improve predictions of genome-wide CRISPR off-target profiles are needed. We focused on the specificity of the traditional CRISPR SpCas9 system for gene knockout. First, we benchmarked 10 available genome-wide off-target cleavage site (OTS) detection techniques with the published OTS detection datasets. Second, taking the datasets generated from OTS detection techniques as the benchmark datasets, we benchmarked 17 available in silico genome-wide OTS prediction tools to evaluate their genome-wide CRISPR off-target prediction performances. Finally, we present the first one-stop integrated Genome-Wide Off-target cleavage Search platform (iGWOS) that was specifically designed for the optimal genome-wide OTS prediction by integrating the available OTS prediction algorithms with an AdaBoost ensemble framework.

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“…Furthermore, the DNA sequence in the cells being investigated can differ substantially from the reference genome used in the computational modeling, potentially resulting in even more false predictions. In recent years, in vitro-based assays [23][24][25][26][27][28] have been developed that allows for experimental detection of Cas9 off-target sites in a particular DNA sample. However, since these methods are based on PCR amplification and short-read sequencing, they have inherent limitations when it comes to detection of Cas9 cleavage in repetitive, low complexity, or AT/GC-rich regions.…”

Section: Introductionmentioning

confidence: 99%

Amplification-free long-read sequencing reveals unforeseen CRISPR-Cas9 off-target activity

et al. 2020

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Background One ongoing concern about CRISPR-Cas9 genome editing is that unspecific guide RNA (gRNA) binding may induce off-target mutations. However, accurate prediction of CRISPR-Cas9 off-target activity is challenging. Here, we present SMRT-OTS and Nano-OTS, two novel, amplification-free, long-read sequencing protocols for detection of gRNA-driven digestion of genomic DNA by Cas9 in vitro. Results The methods are assessed using the human cell line HEK293, re-sequenced at 18x coverage using highly accurate HiFi SMRT reads. SMRT-OTS and Nano-OTS are first applied to three different gRNAs targeting HEK293 genomic DNA, resulting in a set of 55 high-confidence gRNA cleavage sites identified by both methods. Twenty-five of these sites are not reported by off-target prediction software, either because they contain four or more single nucleotide mismatches or insertion/deletion mismatches, as compared with the human reference. Additional experiments reveal that 85% of Cas9 cleavage sites are also found by other in vitro-based methods and that on- and off-target sites are detectable in gene bodies where short-reads fail to uniquely align. Even though SMRT-OTS and Nano-OTS identify several sites with previously validated off-target editing activity in cells, our own CRISPR-Cas9 editing experiments in human fibroblasts do not give rise to detectable off-target mutations at the in vitro-predicted sites. However, indel and structural variation events are enriched at the on-target sites. Conclusions Amplification-free long-read sequencing reveals Cas9 cleavage sites in vitro that would have been difficult to predict using computational tools, including in dark genomic regions inaccessible by short-read sequencing.

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CHANGE-seq reveals genetic and epigenetic effects on CRISPR–Cas9 genome-wide activity

Cited by 171 publications

References 51 publications

Engineered B cells expressing an anti-HIV antibody enable memory retention, isotype switching and clonal expansion

Engineered B cells expressing an anti-HIV antibody enable memory retention, isotype switching and clonal expansion

Benchmarking and integrating genome-wide CRISPR off-target detection and prediction

Amplification-free long-read sequencing reveals unforeseen CRISPR-Cas9 off-target activity

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