The novel insight into the outcomes of CRISPR/Cas9 editing intra- and inter-species

Chang, Jiasong; Chen, Xiaoxu; Zhang, Tong; Wang, Ruolin; Wang, Aoming; Lan, Xinhui; Zhou, Yuyu; Ma, Sanyuan; Xia, Qingyou

doi:10.1016/j.ijbiomac.2020.07.039

Cited by 10 publications

(11 citation statements)

References 45 publications

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“…They presented an accurate predictive model for predicting the outcomes of an edit. 13 Recently, we found that Cas9-induced mutations are coincident in mutation types but are significantly different in indel patterns among species, 14 which prompt the urgency and importance to reveal the editing spectrum of more species other than mammalian. Bombyx mori is an economically important invertebrate and a research model for Lepidoptera insects.…”

Section: Introductionmentioning

confidence: 99%

Deep Sequencing Reveals the Comprehensive CRISPR-Cas9 Editing Spectrum in Bombyx mori

Wang

Chen

et al. 2021

The CRISPR Journal

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Application of the clustered regularly interspaced short palindromic repeats associated 9 (CRISPR-Cas9) technology has revolutionized biology by greatly enhancing the ability to introduce mutations into DNA for research and prospective therapeutic purposes. However, the understanding of Cas9 editing outcomes is still limited. Previously, it was considered that Cas9 introduces stochastic insertions or deletions (indels) at the target site. In the current study, we performed in vivo multiplex editing, deep sequencing, and comprehensive analysis of its editing outcomes in Bombyx mori (B. mori). A total of 31161 editing events from 9 single-guide RNA (sgRNA) sites in 16 individuals were generated and analyzed, and we found that Cas9 introduces mutations with some regularity rather than via stochastic indels. The editing efficiency varies with sgRNA sequences, individuals, and orientation. Small deletions account for the vast majority of mutated sequences, followed by a small fraction of substitutions and insertions. The most likely mutations are deletions between two microhomologous sequences or singlebase deletions at the cleavage site in the absence of microhomologous pairs. Insertions are formed by diverse mechanisms, including direct acquisition of free genomic fragments, duplication of broken ends, replication of adjacent sequences, or random addition of free nucleotides. The above results indicate that the Cas9 editing spectrum is reproducible and predictable. Thus, our findings enable a deeper understanding of Cas9-mediated mutagenesis and better design of genome editing experiments, as well as elucidate the DNA double-strand break repair processes in B. mori.

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Section: Introductionmentioning

confidence: 99%

Deep Sequencing Reveals the Comprehensive CRISPR-Cas9 Editing Spectrum in Bombyx mori

Wang

Chen

et al. 2021

The CRISPR Journal

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“…The tardigrade’s damage suppressor protein (Dsup) halves double-stranded DNA breaks caused by photons in human cells ( 106–108 ). Despite native species potential, research model outcomes are clouded by interspecies differences in the target site sequence structure and DNA repair processes during gene therapy ( 109 ). If a gene proves ultimately incapable of interspecies transfer, expounding upon its mechanism of action may still elucidate novel directions for radioprotection ( 110 ).…”

Section: Discussion: Radioprotective Transgenesmentioning

confidence: 99%

Contemporary biomedical engineering perspective on volitional evolution for human radiotolerance enhancement beyond low-earth orbit

Borg

Baker

2021

Synthetic Biology

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A primary objective of the National Aeronautics and Space Administration (NASA) is expansion of humankind’s presence outside low-Earth orbit, culminating in permanent interplanetary travel and habitation. Having no inherent means of physiological detection or protection against ionizing radiation, humans incur capricious risk when journeying beyond low-Earth orbit for long periods. NASA has made large investments to analyze pathologies from space radiation exposure, emphasizing the importance of characterizing radiation’s physiological effects. Because natural evolution would require many generations to confer resistance against space radiation, immediately pragmatic approaches should be considered. Volitional evolution, defined as humans steering their own heredity, may inevitably retrofit the genome to mitigate resultant pathologies from space radiation exposure. Recently, uniquely radioprotective genes have been identified, conferring local or systemic radiotolerance when overexpressed in vitro and in vivo. Aiding in this process, the CRISPR/Cas9 technique is an inexpensive and reproducible instrument capable of making limited additions and deletions to the genome. Although cohorts can be identified and engineered to protect against radiation, alternative and supplemental strategies should be seriously considered. Advanced propulsion and mild synthetic torpor are perhaps the most likely to be integrated. Interfacing artificial intelligence with genetic engineering using predefined boundary conditions may enable the computational modeling of otherwise overly complex biological networks. The ethical context and boundaries of introducing genetically pioneered humans are considered.

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“…Mutations resulting in deletions were classified into two types: uniquely oriented deletions, which refer to mutations in which the deletion only occurs on one side of the cleavage site, and bidirectional deletions, which refer to mutations in which the deletion occurs at both ends of the cleavage site. In a previous study, researchers revealed that the dominant form of deletion in B. mori was a uniquely oriented deletion consisting primarily of small-fragment deletions [41]. This phenomenon was mainly observed upstream of the cleavage site, unlike in other species.…”

Section: Analysis Of Deletion Patterns In Three Cas9 Proteinsmentioning

confidence: 97%

“…Therefore, it was necessary to compare the editing results of both methods and to determine whether they were similar to the deletion patterns produced by Cas9 editing. In previous research, it has been found that deletions are responsible for the majority of mutations, ranging from 61.50% in M. musculus to 73.11% in Homo sapiens [41]. Therefore, this study primarily compared the differences in deletion patterns among the three species.…”

Section: Analysis Of Deletion Patterns In Three Cas9 Proteinsmentioning

confidence: 98%

High-Throughput Screening of PAM-Flexible Cas9 Variants for Expanded Genome Editing in the Silkworm (Bombyx mori)

Sun,

Zhang,

Lan

et al. 2024

Insects

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Genome editing provides novel opportunities for the precise genome engineering of diverse organisms. Significant progress has been made in the development of genome-editing tools for Bombyx mori (B. mori) in recent years. Among these, CRISPR/Cas9, which is currently the most commonly used system in lepidopteran insects, recognizes NGG protospacer adjacent motif (PAM) sequences within the target locus. However, Cas9 lacks the ability to target all gene loci in B. mori, indicating the need for Cas9 variants with a larger editing range. In this study, we developed a high-throughput screening platform to validate Cas9 variants at all possible recognizable and editable PAM sites for target sequences in B. mori. This platform enabled us to identify PAM sites that can be recognized by both xCas9 3.7 and SpCas9-NG variants in B. mori and to assess their editing efficiency. Cas9 shows PAM sites every 13 base pairs in the genome, whereas xCas9 3.7 and SpCas9-NG have an average distance of 3.4 and 3.6 base pairs, respectively, between two specific targeting sites. Combining the two Cas9 variants could significantly expand the targeting range of the genome, accelerate research on the B. mori genome, and extend the high-throughput rapid screening platform to other insects, particularly those lacking suitable NGG PAM sequences.

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The novel insight into the outcomes of CRISPR/Cas9 editing intra- and inter-species

Cited by 10 publications

References 45 publications

Deep Sequencing Reveals the Comprehensive CRISPR-Cas9 Editing Spectrum in Bombyx mori

Deep Sequencing Reveals the Comprehensive CRISPR-Cas9 Editing Spectrum in Bombyx mori

Contemporary biomedical engineering perspective on volitional evolution for human radiotolerance enhancement beyond low-earth orbit

High-Throughput Screening of PAM-Flexible Cas9 Variants for Expanded Genome Editing in the Silkworm (Bombyx mori)

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