Easy-Prime: a machine learning–based prime editor design tool

Li, Yichao; Chen, Jingjing; Tsai, Shengdar Q.; Cheng, Yong

doi:10.1186/s13059-021-02458-0

Cited by 42 publications

(16 citation statements)

References 31 publications

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“…Depending on the desired edit, researchers often screen pegRNAs with PBS and RT templates of 8-15 nt and 10-74 nt in length, respectively 91 . Given the expense and time required for these optimizations, multiple groups have developed programs for designing pegRNAs [92][93][94][95][96][97][98] . However, only some of these tools can accurately predict well performing pegRNA PBS and RT template lengths for a given target site and edit.…”

Section: Predicting Pegrna Design and Efficiencymentioning

confidence: 99%

“…Although DeepPE optimizes pegRNAs only for one type of edit (a G•C-to-C•G change of a PAM nucleotide), they identified sequence determinants of prime editing efficiency, such as Cas9 nuclease efficiency and GC content of the PBS. Cheng and co-workers also used the dataset from Kim and co-workers to train their own deep learning model, Easy-Prime 95 . These computational tools, as well as models for other types of prime edits that may be developed in the future, have the potential to simplify the successful use of prime editing and broaden its applicability.…”

Section: Predicting Pegrna Design and Efficiencymentioning

confidence: 99%

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Prime editing for precise and highly versatile genome manipulation

2022

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single-stranded DNA (ssDNA) at the target site, triggering deamination by the tethered ssDNA-specific deaminase enzyme. Cytosine base editors (CBEs) and adenine base editors (ABEs) contain deaminases that catalyse C•G-to-T•A and A•T-to-G•C changes, respectively [27][28][29][30][31] . C•G-to-G•C base editors (CGBEs) are similar to CBEs, but stimulate replacement of the deaminated cytosine base with guanine, albeit with lower typical efficiencies and product purities compared to CBEs and ABEs [33][34][35][36] . Compared to Cas nucleases, base editors exhibit substantially greater efficiency with few indel byproducts, and show far fewer undesired consequences of DSBs than nucleases in side-by-side comparisons 19,21,[23][24][25][26]37,38 .Because base editors deaminate within a small window of 4-5 nucleotides (nt) canonically, C or A nucleotides very close to the target C or A can also undergo conversion, resulting in 'bystander editing'. The application of base editors to make changes in the coding sequence of genes usually results in only synonymous mutations within a typical base editing activity window 39 owing to the frequency of transition mutations being silent in the genetic code. Nevertheless, undesired base editing of bystander nucleotides can be challenging to avoid in some cases. Base-editing activity is also restricted by the targeting scope of the Cas domain, which requires the presence of a protospacer adjacent motif (PAM) sequence at a specific distance range (typically 15 ± 2 nt) from the target nucleotide. Moreover, some base editors can induce off-target mutations in DNA and RNA [40][41][42] . Although engineering efforts have mitigated many of these drawbacks [43][44][45][46][47][48][49][50][51] , current base editors can only make six of the 12 possible types of point mutation, leaving many other classes of possible DNA edits, such as insertions, deletions and most transversions, beyond the reach of base editing.Motivated by the need for a precise and highly versatile geneediting technology, prime editing enables all types of DNA substitution, small insertions and small deletions to be installed at targeted sites in the genomes of living cells without directly forming DSBs 52 (Fig. 1c). Prime editing minimally requires a prime editor (PE) protein, which is typically a fusion of a nickase Cas9 (nCas9) and a reverse transcriptase (RT), along with a prime editing guide RNA (pegRNA), which specifies the target site for the edit and contains a programmable RNA template for the desired DNA sequence change. To mediate editing, PEs nick the target site on the genome and extend new DNA sequence from this nick using the pegRNA as a template (Fig. 2). This edited DNA strand is then incorporated into the genome through endogenous cellular processes that can be promoted by also nicking the non-edited strand 52 .Through this mechanism of directly rewriting target DNA, prime editing offers extraordinarily high versatility, editing purity and DNA target specificity in comparison to base editing and HDR with nucl...

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Section: Predicting Pegrna Design and Efficiencymentioning

confidence: 99%

Section: Predicting Pegrna Design and Efficiencymentioning

confidence: 99%

Prime editing for precise and highly versatile genome manipulation

2022

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Section: Main Textmentioning

confidence: 99%

“…A number of computational tools for pegRNA design have been developed [12][13][14][15][16][17][18][19] , but their use for rapid and systematic design of diverse pegRNAs for single, multiplexed, or high-throughput engineering of genetic variants remains limited. Most existing pegRNA design tools are also web-based applications with limited customization capabilities and throughput [12][13][14][15][16][17] . While these applications are very well-suited for designing a handful of pegRNAs, they are often not capable of handling tens of thousands of pegRNA designs at once, which is needed to construct pooled pegRNA libraries for high-throughput screening.…”

Section: Main Textmentioning

confidence: 99%

High throughput evaluation of genetic variants with prime editing sensor libraries

Gould

Sánchez‐Rivera

2022

Preprint

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Prime editing leverages Cas proteins fused to reverse transcription enzymes and prime editing guide RNAs (pegRNAs) to programmably engineer diverse genetic alterations without DNA double strand breaks or exogenous donor templates. Prime editing is powerful, but the process of pegRNA design remains a daunting challenge that limits its widespread adoption by the community. Although a number of computational pegRNA design tools have been developed, none so far contain the right combination of features needed for rapid and systematic design of pegRNAs for variant engineering and high-throughput genetic screens. Here, we describe Prime Editing Guide Generator (PEGG): a computational pipeline for rapid design of pegRNAs and ‘sensor’ pegRNA libraries. PEGG is a user-friendly Python package that generates and visualizes pegRNAs based on a list of input mutations and integrates user-defined properties like variable length of primer binding site and reverse transcription template regions, and outputs ready-to-order pegRNA oligos and libraries containing optimized sequence adapters for pooled cloning. PEGG (including documentation) can be accessed athttps://pegg.readthedocs.io.

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“…Thus, the prime editing guide RNA's design is an important parameter that needs to be optimized for each experimental application to improve editing efficiency while reducing off-target effects. On the basis of the pegRNA design guidance from the original study, several prime editing design tools have become freely available, as summarized in Table 1 [9][10][11][12][13][14][15][16][17][18].…”

Section: Prime Editing Experimental Design Toolsmentioning

confidence: 99%

The potential of CRISPR-Cas9 prime editing for cardiovascular disease research and therapy

Bharucha

Arias

Karakikes

2022

Current Opinion in Cardiology

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Purpose of reviewThe ability to edit any genomic sequence has led to a better understanding of gene function and holds promise for the development of therapies for genetic diseases. This review describes prime editing - the latest CRISPR-Cas9 genome editing technology. Prime editing enables precise and accurate genome editing in terminally differentiated, postmitotic cells like cardiomyocytes, paving the way for therapeutic applications for genetic cardiomyopathies.Recent findingsPrime editing has been used to precisely insert up to 40 bases, create deletions up to 80 base pairs, and can perform all 12 possible transition and transversion base mutations with lower indels and off-target effects than other genome editing methods. The development of several software tools has simplified the experimental design and led to increased efficiency of the process. Improvements in methods for in-vivo delivery of the prime editing components should enable this technology to be used to edit the genome in patients.SummaryPrime editing has the potential to revolutionize the future of biomedical research and transform cardiovascular medicine. Improved understanding of the prime editing process and developments in agent design, efficacy and delivery will benefit scientists and patients and could be an effective way to cure cardiovascular diseases.

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Easy-Prime: a machine learning–based prime editor design tool

Cited by 42 publications

References 31 publications

Prime editing for precise and highly versatile genome manipulation

Prime editing for precise and highly versatile genome manipulation

High throughput evaluation of genetic variants with prime editing sensor libraries

The potential of CRISPR-Cas9 prime editing for cardiovascular disease research and therapy

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