CRISPR prime editing with ribonucleoprotein complexes in zebrafish and primary human cells

Petri, Karl; Zhang, Weiting; Ma, Junyan; Schmidts, Andrea; Lee, Hyunho; Horng, Joy E.; Kim, Daniel Y.; Kurt, Ibrahim Cagri; Clement, Kendell; Hsu, Jonathan Y.; Pinello, Luca; Maus, Marcela V.; Joung, Julia; Yeh, Jing-Ruey Joanna

doi:10.1038/s41587-021-00901-y

Cited by 153 publications

(91 citation statements)

References 19 publications

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“…The highly efficient PEn editing also generated undesired consequences of DSB repair, such as indels, shorter prime edits and longer than intended prime edits which we found to contain additional RT-template integrations. Similar bystander editing was also observed to various extents in the PE2 editing approach 26 . While the presence of the unintended integrations represents a downside of PEn editing, its high robustness and efficiency might be advantageous over the existing methods in situations where a seamless 3' end of the insertion to maintain an open reading frame of the target is not necessary, such as during the correction of frameshift mutations, gene disruption by defined stop codon integration or exon-intron junction editing.…”

Section: Discussionsupporting

confidence: 68%

Harnessing DSB repair to promote efficient homology-dependent and -independent prime editing

Peterka

Akrap

et al. 2021

Preprint

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Prime editing recently emerged as a next-generation approach for precise genome editing. Here we exploit DNA double-strand break (DSB) repair to develop two novel strategies that install precise genomic insertions using an SpCas9 nuclease-based prime editor (PEn). We first demonstrate that PEn coupled to a regular prime editing guide RNA (pegRNA) efficiently promotes short genomic insertions through a homology-dependent DSB repair mechanism. While PEn editing lead to increased levels of by-products, it rescued pegRNAs that performed poorly with a nickase-based prime editor. We also present a small molecule approach that yielded increased product purity of PEn editing. Next, we developed a homology-independent PEn editing strategy by engineering a single primed insertion gRNA (springRNA) which installs genomic insertions at DSBs through the non-homologous end joining pathway (NHEJ). Lastly, we show that PEn-mediated insertions at DSBs prevent Cas9-induced large chromosomal deletions and provide evidence that continuous Cas9-mediated cutting is one of the mechanisms by which Cas9-induced large deletions arise. Altogether, this work expands the current prime editing toolbox by leveraging distinct DNA repair mechanisms including NHEJ, which represents the primary pathway of DSB repair in mammalian cells.

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Section: Discussionsupporting

confidence: 68%

Harnessing DSB repair to promote efficient homology-dependent and -independent prime editing

Peterka

Akrap

et al. 2021

Preprint

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“…While variants of Cas9 with different PAM requirements are rapidly emerging, they are not yet commercially available as recombinant proteins to use in Cas9-gRNA RNP complex injections in zebrafish embryos. Techniques such as base-editing and prime-editing are often seen as the solution to this problem [10,11]. However, the studies describing these techniques have prepared their own recombinant proteins for microinjection, and/or are currently restricted to the injection of in-vitro transcribed mRNA.…”

Section: Introductionmentioning

confidence: 99%

Efficient Generation of Knock-In Zebrafish Models for Inherited Disorders Using CRISPR-Cas9 Ribonucleoprotein Complexes

Vrieze

Bruijn

Reurink

et al. 2021

IJMS

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CRISPR-Cas9-based genome-editing is a highly efficient and cost-effective method to generate zebrafish loss-of-function alleles. However, introducing patient-specific variants into the zebrafish genome with CRISPR-Cas9 remains challenging. Targeting options can be limited by the predetermined genetic context, and the efficiency of the homology-directed DNA repair pathway is relatively low. Here, we illustrate our efficient approach to develop knock-in zebrafish models using two previously variants associated with hereditary sensory deficits. We employ sgRNA-Cas9 ribonucleoprotein (RNP) complexes that are micro-injected into the first cell of fertilized zebrafish eggs together with an asymmetric, single-stranded DNA template containing the variant of interest. The introduction of knock-in events was confirmed by massive parallel sequencing of genomic DNA extracted from a pool of injected embryos. Simultaneous morpholino-induced blocking of a key component of the non-homologous end joining DNA repair pathway, Ku70, improved the knock-in efficiency for one of the targets. Our use of RNP complexes provides an improved knock-in efficiency as compared to previously published studies. Correct knock-in events were identified in 3–8% of alleles, and 30–45% of injected animals had the target variant in their germline. The detailed technical and procedural insights described here provide a valuable framework for the efficient development of knock-in zebrafish models.

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“…Prime editor (PE) 2 is composed of a Cas9-nickase reverse-transcriptase (RT) fusion protein and a prime-editing guide RNA (pegRNA) 1 . PE2 has been shown to induce prime editing in various species and cell types, including human cells [1][2][3][4][5][6][7][8] . However, depending on the target sequence and the used cell type, the efficiency of PE2 is often insufficient 1,2 .…”

mentioning

confidence: 99%

Generation of a more efficient prime editor 2 by addition of the Rad51 DNA-binding domain

et al. 2021

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Although prime editing is a promising genome editing method, the efficiency of prime editor 2 (PE2) is often insufficient. Here we generate a more efficient variant of PE2, named hyPE2, by adding the Rad51 DNA-binding domain. When tested at endogenous sites, hyPE2 shows a median of 1.5- or 1.4- fold (range, 0.99- to 2.6-fold) higher efficiencies than PE2; furthermore, at sites where PE2-induced prime editing is very inefficient (efficiency < 1%), hyPE2 enables prime editing with efficiencies ranging from 1.1% to 2.9% at up to 34% of target sequences, potentially facilitating prime editing applications.

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CRISPR prime editing with ribonucleoprotein complexes in zebrafish and primary human cells

Cited by 153 publications

References 19 publications

Harnessing DSB repair to promote efficient homology-dependent and -independent prime editing

Harnessing DSB repair to promote efficient homology-dependent and -independent prime editing

Efficient Generation of Knock-In Zebrafish Models for Inherited Disorders Using CRISPR-Cas9 Ribonucleoprotein Complexes

Generation of a more efficient prime editor 2 by addition of the Rad51 DNA-binding domain

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