CRISPR-based strategies for targeted transgene knock-in and gene correction

Lau, Cia-Hin; Tin, Chung; Suh, Yousin

doi:10.12703/r/9-20

Cited by 16 publications

(12 citation statements)

References 207 publications

(185 reference statements)

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“…Here we firstly rescued the 140 kb Researchers have made many attempts in CRISPR-based strategies for targeted gene correction and made tremendous advances. The error-prone NHEJ repair pathway is the main DNA repair pathway in non-dividing cells and occurs during the whole cell cycle, whereas the HDR only occurs in S/G2 phase in dividing cells (Lau et al, 2020). The HMEJ strategy is based on both the targeted genomic site and the donor vector with homology arms flanking the recognition sequence of CRISPR/ Cas9 cleaved via CRISPR/Cas9.…”

Section: Discussionmentioning

confidence: 99%

Correction of F8 intron 1 inversion in hemophilia A patient-specific iPSCs by CRISPR/Cas9 mediated gene editing

Wu²,

Xiao³

et al. 2023

Front. Genet.

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Introduction: Hemophilia A (HA) is the most common genetic bleeding disorder caused by mutations in the F8 gene encoding coagulation factor VIII (FVIII). As the second predominant pathogenic mutation in hemophilia A severe patients, F8 Intron one inversion (Inv1) completely splits the F8 gene into two parts and disrupts the F8 transcription, resulting in no FVIII protein production. The part which contains exon 2-exon 26 covers 98% of F8 coding region.Methods: We hypothesized that in situ genetic manipulation of F8 to add a promoter and exon one before the exon two could restore the F8 expression. The donor plasmid included human alpha 1-antitrypsin (hAAT) promoter, exon one and splicing donor site (SD) based on homology-mediated end joining (HMEJ) strategy was targeted addition in hemophilia A patient-derived induced pluripotent stem cell (HA-iPSCs) using CRISPR/Cas9. The iPSCs were differentiated into hepatocyte-like cells (HPLCs).Results: The hAAT promoter and exon one were targeted addition in HA-iPSCs with a high efficiency of 10.19% via HMEJ. The FVIII expression, secretion, and activity were detected in HPLCs derived from gene-targeted iPSCs.Discussion: Thus, we firstly rescued the 140 kb reversion mutation by gene addition of a 975 bp fragment in the HA-iPSCs with Inv1 mutation, providing a promising gene correction strategy for genetic disease with large sequence variants.

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Section: Discussionmentioning

confidence: 99%

Correction of F8 intron 1 inversion in hemophilia A patient-specific iPSCs by CRISPR/Cas9 mediated gene editing

Wu²,

Xiao³

et al. 2023

Front. Genet.

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“…Double-stranded DNA (dsDNA) and single-stranded oligodeoxynucleotides (ssODNs) are the two main formats for HDR donors (8). ssODNs are widely available through commercial DNA synthesis, are non-toxic, and can be delivered to cells in large amounts to maximize knock-in efficiency (39).…”

Section: Additional Design Parameters For Dsdna Vs Ssodn Donorsmentioning

confidence: 99%

“…In the donor, the payload is flanked by sequences homologous to the target site ("homology arms") that can template DNA repair by co-opting endogenous repair pathways (Figure 1A). Because HDR is controllable and precise, it remains the most widely used method for genomic knock-in, although its use is restricted to dividing cells (8). Technical advances are rapidly increasing the efficiency of HDR-based knock-in in key cell types such as human stem cells (9)(10)(11) or primary T cells (12).…”

Section: Introductionmentioning

confidence: 99%

Protospacejam: An Open-Source, Customizable and Web-Accessible Design Platform for Crispr/Cas9 Insertional Knock-In

Peng,

Vangipuram,

Wong

et al. 2023

Preprint

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CRISPR/Cas9-mediated knock-in of DNA sequences enables precise genome engineering for research and therapeutic applications. However, designing effective guide RNAs (gRNAs) and homology-directed repair (HDR) donors remains a bottleneck. Here, we present protoSpaceJAM, an open-source algorithm to automate and optimize gRNA and HDR donor design for CRISPR/Cas9 insertional knock-in experiments. protoSpaceJAM utilizes biological rules to rank gRNAs based on specificity, distance to insertion site, and position relative to regulatory regions. protoSpaceJAM can introduce recoding mutations (silent mutations and mutations in non-coding sequences) in HDR donors to prevent re-cutting and increase knock-in efficiency. Users can customize parameters and design double-stranded or single-stranded donors. We validated protoSpaceJAM’s design rules by demonstrating increased knock-in efficiency with recoding mutations and optimal strand selection for single-stranded donors. An additional module enables the design of genotyping primers for next-generation sequencing of edited alleles. Overall, protoSpaceJAM streamlines and optimizes CRISPR knock-in experimental design in a flexible and modular manner to benefit diverse research and therapeutic applications. protoSpaceJAM is available open-source as an interactive web tool atprotospacejam.czbiohub.orgor as a standalone Python package atgithub.com/czbiohub-sf/protoSpaceJAM.

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“…As we discuss above, CRISPR/Cas-based gene-editing systems have been enhanced and modified by employing various BER enzymes [203,293], made possible due to decades of elegant biochemical analysis of the enzymes in the BER pathway. Overall, BER, and the parallel sub-pathway single-stand break repair (SSBR), are both critical DNA repair pathways that are found in all species and are deemed essential for the resolution of base lesions and DNA single-strand breaks in both the nuclear and mitochondrial genomes.…”

Section: Advances In Ber Biology By Exploiting Gene Editing Technologiesmentioning

confidence: 99%

Exploiting DNA Endonucleases to Advance Mechanisms of DNA Repair

Thompson

Sobol

Prakash

2021

Biology

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The earliest methods of genome editing, such as zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALENs), utilize customizable DNA-binding motifs to target the genome at specific loci. While these approaches provided sequence-specific gene-editing capacity, the laborious process of designing and synthesizing recombinant nucleases to recognize a specific target sequence, combined with limited target choices and poor editing efficiency, ultimately minimized the broad utility of these systems. The discovery of clustered regularly interspaced short palindromic repeat sequences (CRISPR) in Escherichia coli dates to 1987, yet it was another 20 years before CRISPR and the CRISPR-associated (Cas) proteins were identified as part of the microbial adaptive immune system, by targeting phage DNA, to fight bacteriophage reinfection. By 2013, CRISPR/Cas9 systems had been engineered to allow gene editing in mammalian cells. The ease of design, low cytotoxicity, and increased efficiency have made CRISPR/Cas9 and its related systems the designer nucleases of choice for many. In this review, we discuss the various CRISPR systems and their broad utility in genome manipulation. We will explore how CRISPR-controlled modifications have advanced our understanding of the mechanisms of genome stability, using the modulation of DNA repair genes as examples.

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CRISPR-based strategies for targeted transgene knock-in and gene correction

Cited by 16 publications

References 207 publications

Correction of F8 intron 1 inversion in hemophilia A patient-specific iPSCs by CRISPR/Cas9 mediated gene editing

Correction of F8 intron 1 inversion in hemophilia A patient-specific iPSCs by CRISPR/Cas9 mediated gene editing

Protospacejam: An Open-Source, Customizable and Web-Accessible Design Platform for Crispr/Cas9 Insertional Knock-In

Exploiting DNA Endonucleases to Advance Mechanisms of DNA Repair

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