Efficient prime editing in two-cell mouse embryos using PEmbryo

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Abstract

Using transient inhibition of DNA mismatch repair during a permissive stage of development, we demonstrate highly efficient prime editing of mouse embryos with few unwanted, local byproducts (average 58% precise edit frequency, 0.5% on-target error frequency across 13 substitution edits at 8 sites), enabling same-generation phenotyping of founders. Whole-genome sequencing reveals that mismatch repair inhibition increases off-target indels at low-complexity regions in the genome without any obvious phenotype in mice.

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Search-and-replace genome editing without double-strand breaks or donor DNA

Andrew Anzalone, Peyton B. Randolph, Jessie Davis … (2019)

Summary Most genetic variants that contribute to disease 1 are challenging to correct efficiently and without excess byproducts 2–5 . Here we describe prime editing, a versatile and precise genome editing method that directly writes new genetic information into a specified DNA site using a catalytically impaired Cas9 fused to an engineered reverse transcriptase, programmed with a prime editing guide RNA (pegRNA) that both specifies the target site and encodes the desired edit. We performed >175 edits in human cells including targeted insertions, deletions, and all 12 types of point mutations without requiring double-strand breaks or donor DNA templates. We applied prime editing in human cells to correct efficiently and with few byproducts the primary genetic causes of sickle cell disease (requiring a transversion in HBB) and Tay-Sachs disease (requiring a deletion in HEXA), to install a protective transversion in PRNP, and to precisely insert various tags and epitopes into target loci. Four human cell lines and primary post-mitotic mouse cortical neurons support prime editing with varying efficiencies. Prime editing shows higher or similar efficiency and fewer byproducts than homology-directed repair, complementary strengths and weaknesses compared to base editing, and much lower off-target editing than Cas9 nuclease at known Cas9 off-target sites. Prime editing substantially expands the scope and capabilities of genome editing, and in principle can correct up to 89% of known genetic variants associated with human diseases.

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Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases

Sangsu Bae, Jeongbin Park, Jin-Soo Kim (2014)

Summary: The Type II clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system is an adaptive immune response in prokaryotes, protecting host cells against invading phages or plasmids by cleaving these foreign DNA species in a targeted manner. CRISPR/Cas-derived RNA-guided engineered nucleases (RGENs) enable genome editing in cultured cells, animals and plants, but are limited by off-target mutations. Here, we present a novel algorithm termed Cas-OFFinder that searches for potential off-target sites in a given genome or user-defined sequences. Unlike other algorithms currently available for identification of RGEN off-target sites, Cas-OFFinder is not limited by the number of mismatches and allows variations in protospacer-adjacent motif sequences recognized by Cas9, the essential protein component in RGENs. Cas-OFFinder is available as a command-line program or accessible via our website. Availability and implementation: Cas-OFFinder free access at http://www.rgenome.net/cas-offinder. Contact: baesau@snu.ac.kr or jskim01@snu.ac.kr

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CRISPOR: intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens

Jean-Paul Concordet, Maximilian Haeussler (2018)

Abstract CRISPOR.org is a web tool for genome editing experiments with the CRISPR–Cas9 system. It finds guide RNAs in an input sequence and ranks them according to different scores that evaluate potential off-targets in the genome of interest and predict on-target activity. The list of genomes is continuously expanded, with more 150 genomes added in the last two years. CRISPOR tries to provide a comprehensive solution from selection, cloning and expression of guide RNA as well as providing primers needed for testing guide activity and potential off-targets. Recent developments include batch design for genome-wide CRISPR and saturation screens, creating custom oligonucleotides for guide cloning and the design of next generation sequencing primers to test for off-target mutations. CRISPOR is available from http://crispor.org, including the full source code of the website and a stand-alone, command-line version.