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高通量连续进化靶向单嘧啶 PAMs 的紧凑 Cas9 变体。

High-throughput continuous evolution of compact Cas9 variants targeting single-nucleotide-pyrimidine PAMs.

机构信息

Merkin Institute of Transformative Technologies in Healthcare, The Broad Institute of Harvard and MIT, Cambridge, MA, USA.

Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA.

出版信息

Nat Biotechnol. 2023 Jan;41(1):96-107. doi: 10.1038/s41587-022-01410-2. Epub 2022 Sep 8.

DOI:10.1038/s41587-022-01410-2
PMID:36076084
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC9849140/
Abstract

Despite the availability of Cas9 variants with varied protospacer-adjacent motif (PAM) compatibilities, some genomic loci-especially those with pyrimidine-rich PAM sequences-remain inaccessible by high-activity Cas9 proteins. Moreover, broadening PAM sequence compatibility through engineering can increase off-target activity. With directed evolution, we generated four Cas9 variants that together enable targeting of most pyrimidine-rich PAM sequences in the human genome. Using phage-assisted noncontinuous evolution and eVOLVER-supported phage-assisted continuous evolution, we evolved Nme2Cas9, a compact Cas9 variant, into variants that recognize single-nucleotide pyrimidine-PAM sequences. We developed a general selection strategy that requires functional editing with fully specified target protospacers and PAMs. We applied this selection to evolve high-activity variants eNme2-T.1, eNme2-T.2, eNme2-C and eNme2-C.NR. Variants eNme2-T.1 and eNme2-T.2 offer access to NTN PAM sequences with comparable editing efficiencies as existing variants, while eNme2-C and eNme2-C.NR offer less restrictive PAM requirements, comparable or higher activity in a variety of human cell types and lower off-target activity at NCN PAM sequences.

摘要

尽管有不同的原间隔基序（PAM）兼容性的 Cas9 变体可用，但一些基因组位点-特别是那些富含嘧啶的 PAM 序列-仍然无法被高活性 Cas9 蛋白靶向。此外，通过工程拓宽 PAM 序列兼容性会增加脱靶活性。通过定向进化，我们生成了四个 Cas9 变体，它们共同能够靶向人类基因组中大多数富含嘧啶的 PAM 序列。通过噬菌体辅助非连续进化和 EVOLVER 支持的噬菌体辅助连续进化，我们将紧凑的 Cas9 变体 Nme2Cas9 进化为能够识别单核苷酸嘧啶-PAM 序列的变体。我们开发了一种通用的选择策略，该策略需要具有完全指定的目标原间隔基序和 PAMs 的功能编辑。我们将此选择应用于高活性变体 eNme2-T.1、eNme2-T.2、eNme2-C 和 eNme2-C.NR 的进化。变体 eNme2-T.1 和 eNme2-T.2 提供了对 NTN PAM 序列的访问，其编辑效率与现有变体相当，而 eNme2-C 和 eNme2-C.NR 则放宽了 PAM 要求，在多种人类细胞类型中具有相当或更高的活性，并且在 NCN PAM 序列上的脱靶活性更低。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fbc9/9849140/85ccb61080cb/41587_2022_1410_Fig13_ESM.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fbc9/9849140/50357448b753/41587_2022_1410_Fig12_ESM.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fbc9/9849140/85ccb61080cb/41587_2022_1410_Fig13_ESM.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fbc9/9849140/f8aea4dfca50/41587_2022_1410_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fbc9/9849140/0f63d576acbd/41587_2022_1410_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fbc9/9849140/df2826dbfdc6/41587_2022_1410_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fbc9/9849140/5bc9361ec4e6/41587_2022_1410_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fbc9/9849140/b26b1c2c4a4b/41587_2022_1410_Fig5_ESM.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fbc9/9849140/ba66ca391c37/41587_2022_1410_Fig6_ESM.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fbc9/9849140/07099bab43b2/41587_2022_1410_Fig7_ESM.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fbc9/9849140/a22bcf02e149/41587_2022_1410_Fig8_ESM.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fbc9/9849140/98cc404a5ebb/41587_2022_1410_Fig9_ESM.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fbc9/9849140/d67d6192c8f5/41587_2022_1410_Fig10_ESM.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fbc9/9849140/641bd0bce0c8/41587_2022_1410_Fig11_ESM.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fbc9/9849140/50357448b753/41587_2022_1410_Fig12_ESM.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fbc9/9849140/85ccb61080cb/41587_2022_1410_Fig13_ESM.jpg

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