Several ABEs by the group led by David

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Several ABEs by the group led by David

Several generations of of ABEs were developed, so that
ultimately seventh generation improved ABEs included the
following four
ABEs: ABE 6.3, ABE 7.8, ABE 7.9
and ABE 7.10911. Of these, ABE7.10 was the most active editor, with
high level of editing efficiency (53%). The editing window, however, was still
4-7 in ABE7.10 and 4-9 in the other three seventh generation ABEs.  These ABEs did not display any significant A to non-G conversion at
target loci,
because the removal of inosine through BSE from DNA is not as common as that of
uracil (U)9 due
to the presence of UNG11. ABEs also
performed better than many BEs in terms of off-target editing and
frequency of indels
produced during editing. In an actual study, ABE7.10 modified only
4/12 off-targets with a frequency of 1.2% indels, in comparison with
9/12 known
off-targets with a 14% indel rate in other BEs.

 

Base editing in RNA
and ‘REPAIR’ technology

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In
October, 2017, along with the report of ABEs by the group led by David Lui11,
base editors were also developed for editing of RNA transcripts. These RNA base
editors were developed by another group led by Feng Zhang24 of the
Broad Institute, who has also been visible in recent years due to CRISPR patent
battle25.. In most studies on
base editing, target site occurs in
genomic DNA, but this technology has been limited by the requirement of
canonical PAM-NGG site at the target locus, although base editors for sites
with non-canonical PAM were also developed (discussed earlier). Keeping this in
view, RNA base editors were developed using
the programmable type VI CRISPR-associated RNA-guided RNase Cas13b; the
technology has been described as REPAIR RNA editing for programmable A to I
(G) replacement12. The naturally occurring ADAR (adenosine
deaminase acting on RNA) was used with disabled Cas13 (dCas13) and guide RNA
(gRNA) for endogenous A®I editing at specific
transcript targets in mammalian cells. The specificity of dCas13b was further improved
through mutagenesis to generate REPAIRv1 and then REPAIRv2, which had very high level of specificity. Hopefully,
REPAIRv2 or other improved form of REPAIR will be utilized in future for a
variety of purposes, particularly for teansient alteration in the transcripts, where s
permanent alteration of the genome is not required (e.g., temporary relief from
a disease).

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