Engineered miniature CRISPR-Cas system for mammalian genome regulation and editing

Xu, Xiaoshu; Chemparathy, Augustine; Zeng, Leiping; Kempton, H.; Shang, Stephen; Nakamura, Morimasa; Qi, Lei S.

doi:10.1016/j.molcel.2021.08.008

Cited by 251 publications

(224 citation statements)

References 42 publications

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“…This discussion highlights the potential of small Cas orthologs such as CasMINI that would allow an all-in-one packaging approach of gRNA, base editor and Cas protein in a single AAV vector [ 49 ]. In addition to being half the size of SpCas9 (529aa), CasMINI [ 20 ] has the additional benefit of a small editing window that would greatly limit bystander mutations.…”

Section: Discussionmentioning

confidence: 99%

“…Recently, a mutant variant of the Cas12f family, which is less than half the size of SpCas9, was reported to function in mammalian cell line. CasMINI has the same PAM as LbCas12a, but features a very restrictive editing window, which makes the search for appropriate PAM sites more challenging but limits the possibility of bystander edits [ 20 ].…”

Section: Introductionmentioning

confidence: 99%

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

Bellingrath

McClements

Kaukonen

et al. 2021

Genes

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Bellingrath

McClements

Kaukonen

et al. 2021

Genes

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“…While pDNA can be transfected to dividing cells to some extent and used for the creation of stable cell lines via FACS sorting, delivery to neurons is near-impossible with pDNA. Since the gene editing field is rapidly developing, researchers have found ways to overcome the size limitations, including the recently introduced downsized Cas9 and BE, which fit in a single rAAV, speeding up future gene editing efforts [44].…”

Section: Permanent Gene Therapies That Alter the Genomementioning

confidence: 99%

Gene Therapies for Monogenic Autism Spectrum Disorders

Weuring

Geerligs

Koeleman

2021

Genes

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Novel genome editing and transient gene therapies have been developed the past ten years, resulting in the first in-human clinical trials for monogenic disorders. Syndromic autism spectrum disorders can be caused by mutations in a single gene. Given the monogenic aspect and severity of syndromic ASD, it is an ideal candidate for gene therapies. Here, we selected 11 monogenic ASD syndromes, validated by animal models, and reviewed current gene therapies for each syndrome. Given the wide variety and novelty of some forms of gene therapy, the best possible option must be decided based on the gene and mutation.

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“…Obligated mobile element-guided activity (OMEGA) is another class of transposon-encoded RGENs with a reduced size (∼400 amino acids) ( Altae-Tran et al, 2021 ). Miniature CRISPR associated RGENs such as Cas12f are also being explored for gene editing capability and engineered for improved efficiency ( Bigelyte et al, 2021 ; Xu et al, 2021 ). The discovery and optimization of these smaller genome editing tools may facilitate delivery with germline infecting PSVs such as TRV or BSMV.…”

Section: Virus Induced Genome Editingmentioning

confidence: 99%

Non-GM Genome Editing Approaches in Crops

2021

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CRISPR/Cas-based genome editing technologies have the potential to fast-track large-scale crop breeding programs. However, the rigid cell wall limits the delivery of CRISPR/Cas components into plant cells, decreasing genome editing efficiency. Established methods, such as Agrobacterium tumefaciens-mediated or biolistic transformation have been used to integrate genetic cassettes containing CRISPR components into the plant genome. Although efficient, these methods pose several problems, including 1) The transformation process requires laborious and time-consuming tissue culture and regeneration steps; 2) many crop species and elite varieties are recalcitrant to transformation; 3) The segregation of transgenes in vegetatively propagated or highly heterozygous crops, such as pineapple, is either difficult or impossible; and 4) The production of a genetically modified first generation can lead to public controversy and onerous government regulations. The development of transgene-free genome editing technologies can address many problems associated with transgenic-based approaches. Transgene-free genome editing have been achieved through the delivery of preassembled CRISPR/Cas ribonucleoproteins, although its application is limited. The use of viral vectors for delivery of CRISPR/Cas components has recently emerged as a powerful alternative but it requires further exploration. In this review, we discuss the different strategies, principles, applications, and future directions of transgene-free genome editing methods.

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Engineered miniature CRISPR-Cas system for mammalian genome regulation and editing

Cited by 251 publications

References 42 publications

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

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

Gene Therapies for Monogenic Autism Spectrum Disorders

Non-GM Genome Editing Approaches in Crops

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