A new method of prime editing
Elena Kleshchenko, PCR.news
The method of primed genome editing (prime editing) developed at the Brody Institute in 2019. It allows you to make fairly extensive and precise corrections to the DNA. The editing system includes a catalytically weakened Cas9 nuclease crosslinked with reverse transcriptase (RT) and a special guide RNA — prime editing guide RNA (pegRNA), which not only directs the nuclease to the desired DNA site, but also serves as a matrix for "rewriting" this site. The design guided by pegRNA sits on the DNA and cuts one DNA chain. The free end of the pegRNA containing the primer sequence binds to the free end of the DNA, and then the reverse transcriptase completes the strand at the incision site using pegRNA as a matrix. A new DNA fragment takes the place of the original sequence, and then cellular DNA repair mechanisms are activated. Since the cut thread is usually repaired, sometimes another CRISPR construct is used, which cuts the second, unedited thread so that it "rewrites" the first one.
Prime editing systems have shown high accuracy and efficiency, but the size of the protein structure combining two enzymes is very large, and this creates difficulties in delivering coding sequences in viral vectors. Now a group of researchers from the USA and Germany has found that it is not necessary to covalently cross-link two enzymes for primed editing. They investigated the construction of the PE2 — nicase Cas9 bacteria on cells in culture Streptococcus pyogenes (nSpCas9), to the C-end of which the reverse transcriptase of the Moloney mouse leukemia virus (MMLV-RT) is sewn. Various configurations were compared, for example, attaching MMLV-RT to the N-end or placing it inside the Cas9 coding sequence. It turned out that the unrelated proteins nSpCas9 and MMLV-RT function in human cells as efficiently as other variants of PE2 — the editing accuracy remained high for both substitutions and insertions and deletions. The coding sequence of nSpCas9 can be delivered in one vector, and RNA and reverse transcriptase can be delivered in the other.
This system, which the authors called Split-PE, can be used to quickly find and evaluate new options for prime editors. This, in particular, will expand the set of reverse transcriptases used. The authors tested six shortened mutant forms of MMLV-RT and found a compact variant, no less active than the parent one, and also evaluated the potential of Marathon-RT reverse transcriptase from Eubacterium rectale, which is often used in in vitro experiments.
The researchers also showed that PE2 can be divided not between the nicase and reverse transcriptase, but inside the nicase, and provided with inteins (sites necessary for protein splicing). Such a construction can also be delivered to the cell in two adeno-associated vectors, and as a result, a functional protein will be restored.
Article by Gruenewald et al. Engineered CRISPR prime editors with compact, untethered reverse transcriptases published in the journal Nature Biotechnology.
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